KR102624979B1 - B4GALT1 variants and their uses - Google Patents

Abstract

Variant B4GALT1 genomes, mRNA and cDNA nucleic acid molecules and polypeptides, methods for detecting the presence of such molecules, methods for regulating endogenous B4GALT1 genomes, mRNA and cDNA nucleic acid molecules and polypeptides, variant B4GALT1 genomes, mRNA and cDNA nucleic acid molecules and Provided herein are methods for determining the risk of developing a cardiovascular condition and methods for treating a cardiovascular condition by detecting the presence or absence of a polypeptide.

Description

B4GALT1 variants and their uses

Note on government deeds

This invention was made with government support under HL121007 awarded by the National Institutes of Health. The Government has certain rights in the invention.

Note on sequence listing

This application contains a sequence listing submitted electronically as a text file with file name 18923800202SEQ, created on June 4, 2018, and having a size of 161 KB. The sequence listing is incorporated herein by reference.

Technology field

The present disclosure discloses variant B4GALT1 (beta-1,4-galactosyltransferase 1) genomes, mRNA and cDNA nucleic acid molecules and polypeptides, methods for detecting the presence of such molecules, and modulating endogenous B4GALT1 genomes, mRNA and cDNA nucleic acid molecules and polypeptides. Methods for determining the risk of developing cardiovascular conditions by detecting the presence or absence of the variant B4GALT1 genome, mRNA and cDNA nucleic acid molecules and polypeptides, and methods for treating cardiovascular conditions are provided.

Various publications are cited throughout this specification, including patents, published applications, registration numbers, technical papers, and academic papers. Each cited publication is herein incorporated by reference in its entirety and for all purposes.

Beta-1,4-galactosyltransferase 1 ( B4GALT1 ) encodes a type II membrane-bound glycoprotein that plays a role in the biosynthesis of different glycoconjugates and saccharide structures. , is a member of the 4-galactosyltransferase gene family. The enzyme encoded by B4GALT1 plays an important role in the processing of N-linked oligosaccharide moieties in glycoproteins, and protein-linked sugar chains usually regulate the biological functions of glycoproteins. Therefore, impaired B4GALT1 activity has the potential to alter the structure of all glycoproteins containing N-linked oligosaccharides. The long form of the B4GALT1 enzyme is localized to the trans-Golgi, where it attaches galactosyl residues to N-acetylglucosamine residues during biosynthetic processing into complex-type N-linked oligosaccharides of high-mannose. Deliver. Because the addition of galactosyl residues is a prerequisite for the addition of sialic acid, defects in B4GALT1 may exert an indirect effect, blocking the addition of sialic acid residues and thus altering the half-life of the plasma glycoprotein. It has been reported that defects in glycosylation impair intracellular trafficking of various glycoproteins, including the LDL receptor. Additionally, structural abnormalities in N-linked oligosaccharides have the potential to alter protein folding, which in turn may alter the function of the glycoprotein and its secretion. Many proteins contain N-linked glycosylation, including cell surface receptors (e.g., LDL receptor and insulin receptor) as well as various circulating plasma proteins (e.g., apolipoprotein B and fibrinogen). Patients with genetic diseases due to homozygosity for protein-truncating mutations in the B4GALT1 gene have been reported. One such patient had a severe phenotype characterized by a) severe neurodevelopmental abnormalities (including hydrocephalus), b) myopathy, and c) blood coagulation abnormalities. As expected, oligosaccharides derived from cyclic transferrin lacked galactose and sialic acid residues. Two additional patients with the same gene defect appeared to have a milder phenotype, characterized by coagulation disturbances, hepatic dysfunction and dysmorphic features.

Cardiovascular disease is a leading cause of death in the United States and other Western countries. Major risk factors for atherothrombotic cardiovascular disease, such as stroke and myocardial infarction, include increased blood cholesterol and a tendency for blood clots. Many proteins involved in lipid metabolism and coagulation are glucosylated and therefore regulated by B4GALT1 . Knowledge of the genetic factors underlying the onset and progression of cardiovascular conditions can improve risk stratification and provide a foundation for novel treatment strategies.

The present disclosure provides nucleic acid molecules comprising a nucleic acid sequence that is at least about 90% identical to a B4GALT1 variant genomic sequence (including the SNP designated rs551564683 ), provided that the nucleic acid sequence also differs from position 352 of the full-length/mature B4GALT1 polypeptide. Contains a nucleotide encoding serine at the corresponding position.

The present disclosure also provides a nucleic acid molecule comprising a nucleic acid sequence that is at least about 90% identical to a B4GALT1 variant mRNA sequence (including the SNP designated rs551564683 ), provided that the nucleic acid sequence also includes a nucleic acid sequence at position 352 of the full-length/mature B4GALT1 polypeptide. encodes serine at the corresponding position.

The present disclosure also provides a cDNA molecule encoding a B4GALT1 polypeptide comprising a nucleic acid sequence at least about 90% identical to a B4GALT1 variant cDNA sequence (including the SNP designated rs551564683 ), provided that the nucleic acid sequence also represents the full-length/mature B4GALT1 It encodes serine at the position corresponding to position 352 in the polypeptide.

The present disclosure also provides vectors or exogenous donor sequences comprising any one or more of these nucleic acid molecules.

The present disclosure also provides an isolated polypeptide comprising an amino acid sequence that is at least about 90% identical to a B4GALT1 polypeptide with a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide.

The present disclosure also provides host cells comprising any one or more of these nucleic acid molecules operably linked to a heterologous promoter that is active in the host cell.

The present disclosure also provides steps in which the nucleic acid molecule is expressed by culturing a host cell containing a nucleic acid molecule encoding a B4GALT1 polypeptide, wherein the nucleic acid molecule is operably linked to a heterologous promoter active in the host cell, and the isolated polypeptide. A method for producing B4GALT1 polypeptide by recovering the peptide is provided.

The present disclosure also provides compositions comprising such nucleic acid molecules or polypeptides and a carrier to increase their stability.

The present disclosure also provides a method for detecting the presence or absence of a B4GALT1 variant nucleic acid molecule (comprising a SNP designated as rs551564683 ) in a human subject, comprising performing the assay on a biological sample from the human subject to determine the biological and determining whether the nucleic acid molecules in the sample comprise a nucleic acid sequence encoding a variant B4GALT1 polypeptide with a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide.

The present disclosure also provides a method for detecting the presence of a variant B4GALT1 polypeptide with a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide in a human subject, the method comprising: and performing an assay to determine the presence of the variant B4GALT1 polypeptide.

The present disclosure also provides a method of determining the susceptibility of a human subject to the development of a cardiovascular condition, comprising: a) performing the assay on a biological sample from the human subject to determine whether the nucleic acid molecule in the biological sample is full-length/mature; determining whether it comprises a nucleic acid sequence encoding a variant B4GALT1 polypeptide having a serine at a position corresponding to position 352 in the B4GALT1 polypeptide; and b) a nucleic acid molecule comprising a nucleic acid sequence encoding a variant B4GALT1 polypeptide having a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide is detected in the biological sample, predisposing the human subject to the development of a cardiovascular condition. classified as having a reduced risk for or if no nucleic acid molecule comprising a nucleic acid sequence encoding a variant B4GALT1 polypeptide with a serine at position 352 in the full-length/mature B4GALT1 polypeptide is detected in the biological sample. , comprising classifying the human subject as having an increased risk for developing a cardiovascular condition.

The disclosure also provides a method of determining the susceptibility of a human subject to the development of a cardiovascular condition, the method comprising: a) performing the assay on a biological sample from the human subject to determine whether the B4GALT1 polypeptide in the biological sample is 352; determining whether a serine is included at a position corresponding to the position; and b) if a B4GALT1 polypeptide with a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide is detected in the biological sample, the human subject is classified as having a reduced risk for developing a cardiovascular condition, or If a B4GALT1 polypeptide with a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide is not detected in the biological sample, classifying the human subject as having an increased risk for developing a cardiovascular condition. .

The present disclosure also provides guide RNA molecules effective for directing a Cas enzyme to bind to or cleave an endogenous B4GALT1 gene, wherein the guide RNA comprises a position corresponding to positions 53575 to 53577 of the wild-type B4GALT1 gene, or and a DNA-targeting segment that hybridizes to a guide RNA recognition sequence within the endogenous B4GALT1 gene adjacent thereto (e.g., within a certain number of nucleotides, as discussed below).

The present disclosure also provides a method of modifying the endogenous B4GALT1 gene in a cell, comprising modifying the genome of the cell with a) a Cas protein; and b) contacting a guide RNA that forms a complex with the Cas protein and hybridizes to a guide RNA recognition sequence in the endogenous B4GALT1 gene, wherein the guide RNA recognition sequence corresponds to positions 53575 to 53577 of the wild-type B4GALT1 gene. Containing or adjacent to the site (e.g., within a certain number of nucleotides, as discussed below), the Cas protein cleaves the endogenous B4GALT1 gene.

The present disclosure also provides a method of modifying the endogenous B4GALT1 gene in a cell, comprising modifying the genome of the cell with a) a Cas protein; and b) contacting a first guide RNA that forms a complex with the Cas protein and hybridizes to a first guide RNA recognition sequence in the endogenous B4GALT1 gene, wherein the first guide RNA recognition sequence is the start codon for the B4GALT1 gene. or is within about 1,000 nucleotides of the start codon, wherein the Cas protein cleaves the endogenous B4GALT1 gene or alters its expression.

The present disclosure also provides a method of transforming a cell, comprising introducing an expression vector into the cell, wherein the expression vector contains a serine expression at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide. It includes a recombinant B4GALT1 gene comprising a nucleotide sequence encoding a B4GALT1 polypeptide.

The present disclosure also provides a method of transforming a cell, comprising introducing an expression vector into the cell, wherein the expression vector contains a serine expression at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide. A nucleic acid molecule encoding a polypeptide that is at least about 90% identical to a B4GALT1 polypeptide having a

The present disclosure also provides a method of modifying a cell, comprising introducing into the cell a polypeptide or fragment thereof, wherein the polypeptide corresponds to position 352 in the full-length/mature B4GALT1 polypeptide. is at least 90% identical to a B4GALT1 polypeptide with a serine at position, wherein the polypeptide also comprises a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide.

The present disclosure also provides a method of treating a subject who is not a carrier of a B4GALT1 variant nucleic acid molecule or polypeptide (including the SNP designated as rs551564683 ) and has, or is susceptible to developing, a cardiovascular condition, which method comprises: ) Cas protein or nucleic acid encoding Cas protein; b) a guide RNA or a nucleic acid encoding a guide RNA, wherein the guide RNA forms a complex with a Cas protein and hybridizes to a guide RNA recognition sequence in the endogenous B4GALT1 gene, and the guide RNA recognition sequence is located at positions 53575 to 53577 of the wild-type B4GALT1 gene. A guide RNA, or a nucleic acid encoding a guide RNA, comprising or adjacent to a position corresponding to the position; and c) a 5' homology arm that hybridizes to the target sequence 5' at a position corresponding to positions 53575 to 53577 of the wild-type B4GALT1 gene, hybridizing to the target sequence 3' at a position corresponding to positions 53575 to 53577 of the wild-type B4GALT1 gene. A nucleotide sequence encoding a B4GALT1 polypeptide having a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide flanked by the 3' homology arm and the 5' homology arm and the 3' homology arm. Introducing an exogenous donor sequence comprising a nucleic acid insert, wherein the Cas protein cleaves the endogenous B4GALT1 gene within a cell in the subject, the exogenous donor sequence recombines with the endogenous B4GALT1 gene within the cell, and the exogenous donor sequence and Upon recombination with the endogenous B4GALT1 gene, serine is inserted at the nucleotide corresponding to positions 53575 to 53577 of the wild-type B4GALT1 gene.

The present disclosure also provides a method of treating a subject who is not a carrier of a B4GALT1 variant nucleic acid molecule or polypeptide (including the SNP designated as rs551564683 ) and has, or is susceptible to developing, a cardiovascular condition, which method comprises: ) Cas protein or nucleic acid encoding Cas protein; b) a first guide RNA or a nucleic acid encoding a first guide RNA, wherein the first guide RNA forms a complex with a Cas protein and hybridizes to a first guide RNA recognition sequence in the endogenous B4GALT1 gene, wherein the first guide RNA recognizes The sequence includes a first guide RNA or a nucleic acid encoding a first guide RNA that includes the start codon for the endogenous B4GALT1 gene or is within about 1,000 nucleotides of the start codon; c) introducing an expression vector comprising a recombinant B4GALT1 gene comprising a nucleotide sequence encoding a B4GALT1 polypeptide with a serine at position 352 corresponding to position 352 in the full-length/mature B4GALT1 polypeptide, wherein the Cas protein cleaves or alters the expression of the endogenous B4GALT1 gene in a cell in the subject, and the expression vector expresses the recombinant B4GALT1 gene in a cell in the subject.

The present disclosure also provides a method of treating a subject who is not a carrier of a B4GALT1 variant nucleic acid molecule or polypeptide (including the SNP designated as rs551564683 ) and has or is susceptible to developing a cardiovascular condition, comprising: , introducing antisense DNA, RNA, siRNA or shRNA that hybridizes to a sequence within the endogenous B4GALT1 gene and reduces expression of the B4GALT1 polypeptide in cells within the subject.

The present disclosure also provides a method of treating a subject who is not a carrier of a B4GALT1 variant nucleic acid molecule or polypeptide (including the SNP designated as rs551564683 ) and has, or is susceptible to developing, a cardiovascular condition, comprising: Introducing an expression vector, wherein the expression vector comprises a recombinant B4GALT1 gene comprising a nucleotide sequence encoding a B4GALT1 polypeptide with a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide, wherein the expression vector expresses the recombinant B4GALT1 gene in cells within the subject.

The present disclosure also provides a method of treating a subject who is not a carrier of a B4GALT1 variant nucleic acid molecule or polypeptide (including the SNP designated as rs551564683 ) and has, or is susceptible to developing, a cardiovascular condition, comprising: Introducing an expression vector, wherein the expression vector comprises a nucleic acid molecule encoding a B4GALT1 polypeptide with a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide, wherein the expression vector is The cells express nucleic acid encoding the B4GALT1 polypeptide.

The present disclosure also provides a method of treating a subject who is not a carrier of a B4GALT1 variant nucleic acid molecule or polypeptide (including the SNP designated as rs551564683 ) and has, or is susceptible to developing, a cardiovascular condition, comprising: Introducing an mRNA, wherein the mRNA encodes a B4GALT1 polypeptide with a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide, and wherein the mRNA expresses the B4GALT1 polypeptide in a cell in the subject. .

The present disclosure also provides a method of treating a subject who is not a carrier of a B4GALT1 variant nucleic acid molecule or polypeptide (including the SNP designated as rs551564683 ) and has, or is susceptible to developing, a cardiovascular condition, comprising: Introducing a B4GALT1 polypeptide or fragment thereof with a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide.

In any of the methods described or exemplified herein, the cardiovascular condition may include levels of one or more serum lipids that increase atherosclerotic risk. Serum lipids include cholesterol, LDL, HDL, triglycerides, HDL-cholesterol, and non-HDL cholesterol or their subclasses (e.g., HDL2, HDL2a, HDL2b, HDL2c, HDL3, HDL3a, HDL3b, HDL3c, HDL3d, LDL1). , LDL2, LDL3, lipoprotein A, Lpa1, Lpa1, Lpa3, Lpa4 or Lpa5). Cardiovascular conditions may include increased levels of coronary artery calcification. Cardiovascular conditions may include increased levels of pericardial fat. Cardiovascular conditions may include atherothrombotic conditions. Atherothrombotic conditions may involve increased levels of fibrinogen. Atherothrombotic conditions may include fibrinogen-mediated blood clots. Cardiovascular conditions may involve increased levels of fibrinogen. Cardiovascular conditions may include fibrinogen-mediated blood clots. Cardiovascular conditions may include blood clots that form from involvement of fibrinogen activity. Fibrinogen-mediated blood clots, or blood clots formed from involvement of fibrinogen activity, can be present within any vein or artery within the body.

Figure 1 shows the results of a representative genome-wide association of variant B4GALT1 and LDL.
Figure 2 is a diagram showing the results of representative TOPMed WGS association of variant B4GALT1 and LDL.
Figure 3 shows the results of a representative haplotype structure of the top B4GALT1 -linked SNPs.
Figure 4 is a diagram showing the association between the variant B4GALT1 gene and LDL in the Amish identified by exome sequencing.
Figure 5 is a diagram showing that the frequency of the variant B4GALT1 gene is more than 1000 times more abundant in the Amish.
Figure 6 shows the association between B4GALT1 Asn352Ser and reduced serum lipids.
Figure 7 shows the high degree of association between B4GALT1 Asn352Ser and decreased serum lipids and increased AST.
Figure 8 shows the association of B4GALT1 Asn352Ser with all lipid subclasses.
Figure 9 shows the association between B4GALT1 Asn352Ser and reduced fibrinogen levels.
Figure 10 shows the reduced B4GALT1 transcript within 5 days post fertilization of zebrafish larvae injected with antisense morpholino oligonucleotide at the indicated concentration.
Figure 11 shows diagnostic markers of antisense morpholino oligonucleotide off-target effects within 5 days after fertilization of zebrafish Larva injected with antisense morpholino oligonucleotide at the indicated concentration.
Figure 12 is a diagram showing the average LDL concentration in 100 homogenates 5 days after fertilization of zebrafish Lava according to the experiment.
Figure 13 shows rescue of the LDL-c phenotype by co-expression of 50 pg of human B4GALT1 mRNA in zebrafish.
Figure 14 shows the results of genetic association between B4GALT1 N352S and LDL using targeted genotyping.
Figure 15 is a confocal microscopy image of subcellular localization of Flag-352Asn or Flag-352Ser.
Figure 16 is a confocal microscopy image of the intracellular localization of endogenous B4GALT1, Flag-352Asn and Flag-352Se in relation to the trans Golgi Network marker TGN46.
Figure 17 (Panels A and B) shows the effect of 352Ser on steady-state levels of B4GALT1 protein; (Panel A) COS7 cells expressing 352Asn or 352Ser Flag tag protein fusions with free EGFP; and (Panel B) mRNA expression levels for the B4GALT1 gene determined by RT-qPCR analysis.
Figure 18 (Panels A, B and C) shows the effect of the 352Ser mutation on activity; (Panels A and B) COS7 cells expressing 352Asn or 352Ser Flag tag protein fusions and analyzed by Western blot for B4GALT1 or Flag; (Panel C) B4GALT1 activity in immunoprecipitates.
Figure 19 is a diagram showing the tri-sialo/di-oligo ratio by B4GALT1 N352S genotype group.
Figure 20 shows representative HILIC-FLR-MS spectra of N-glycan analysis of glycoproteins from matched pairs of minor (SS) and major (NN) homozygotes of B4GALT1 N352S.

As mentioned herein, sequencing studies have shown that the full-length/mature gene instead of asparagine is present in approximately 11% to 12% of Old Order Amish (OOA) individuals (alternative allele frequency = 6%). We identify variants of B4GALT1 that have a serine at the position corresponding to position 352 in the B4GALT1 polypeptide, and are extremely rare in the general population. This mutation changes asparagine to serine at position 352 (N352S) in the 398 amino acid long human protein or at position 311 in the short isoform. Variant B4GALT1 has lower levels of low-density lipoprotein cholesterol (LDL), total cholesterol and fibrinogen, and eGFR, increased levels of aspartate transaminase (AST) (but not alanine transaminase (ALT)), and creatine. It was found to be associated with serum levels of kinase and creatinine, their expression in muscle tissue (but not the liver or red blood cells), and a decrease in basophils. The N352S variant is believed to be protective against one or more cardiovascular conditions. It is further believed that B4GALT1 , including variant status, may be used to diagnose a patient's risk of developing cardiovascular conditions.

The phrase "corresponding to", when used in connection with the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of residues in a specified standard sequence when comparing a given amino acid or polynucleotide sequence to a standard sequence (as used herein, a standard sequence is (wild type/full length) is a polynucleotide (gDNA sequence, mRNA sequence, cDNA sequence) or polypeptide of B4GALT1 . That is, the residue number or residue position of a given polymer is assigned relative to a standard sequence rather than by the actual number position of the residue within a given amino acid or polynucleotide sequence. For example, a given amino acid sequence can be aligned to a standard sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, the numbering of residues in a given amino acid or polynucleotide sequence is performed relative to the standard sequence to which it is aligned, although gaps exist.

As used herein, the singular forms include the plural, unless the context clearly dictates otherwise.

As used herein, and unless otherwise clear from the context, “about” includes values within the standard error of measurement (e.g., SEM) of the stated value.

As used herein, the term "and/or" refers to or includes any and all possible combinations of one or more of the associated listed items, as well as lack of combinations ("or") when alternatively interpreted. do.

As used herein, the terms “comprising” or “including” mean that one or more of the mentioned elements may include other elements not specifically mentioned. For example, a composition “comprising” or “comprising” a protein may contain the protein alone or in combination with other ingredients. The conjunction "consisting essentially of" means that the scope of the claim shall be construed to include the specified elements recited in the claim and without materially affecting the basic and novel feature(s) of the subject matter of the claimed invention. it means. Accordingly, the term “consisting essentially of” when used in the claims of this disclosure is not intended to be construed as equivalent to “comprising.”

As used herein, “optional” or “optionally” means that a subsequently described event or circumstance may or may not occur and such description includes instances in which the event or circumstance occurs and instances in which it does not occur. means to include.

As used herein, “or” refers to any one member of a particular list, and also includes any combination of members of that list.

The specification of a range of values includes all integers within or delimiting the range (including both endpoint values), and all subranges defined by integers within the range.

It should be recognized that certain features of the disclosure that are described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the disclosure that are described in the context of a single embodiment for the sake of simplicity may also be provided separately or in any suitable subcombination.

The present disclosure provides isolated B4GALT1 genomic and mRNA variants, B4GALT1 cDNA variants, or any complement thereof, and isolated B4GALT1 polypeptide variants. These variants are associated with increased levels of serum lipids, and increased levels of fibrinogen, coronary artery calcification, coronary artery disease (CAD), and increased levels of aspartate aminotransferase (AST) but not alanine transaminase (ALT). It is believed to be associated with a reduced risk of developing a variety of cardiovascular conditions, including but not limited to: Without wishing to be bound by any theory, it is believed that this B4GALT1 variant is associated with expression in muscle tissue but not in the liver or red blood cells, as evidenced by experimentally observed increased levels of AST but not ALT. Also provided herein are compositions comprising B4GALT1 genomic and mRNA variants, B4GALT1 cDNA variants, and isolated B4GALT1 polypeptide variants. Nucleic acid molecules that hybridize to B4GALT1 genomic and mRNA variants and B4GALT1 cDNA variants are also provided herein. The disclosure also provides vectors and cells comprising B4GALT1 genomic and mRNA variants, B4GALT1 cDNA variants, and B4GALT1 polypeptide variants.

The disclosure also provides methods for detecting the presence and/or levels of genomic and/or mRNA variants, B4GALT1 cDNA variants, or complements thereof, and/or B4GALT1 polypeptide variants in a biological sample. Also provided are methods of determining a subject's susceptibility to developing a cardiovascular condition, and methods of diagnosing a subject with a cardiovascular condition or at risk for a cardiovascular condition. There is also a method of transforming a cell by using any combination of a nuclease agonist, an exogenous donor sequence, a transcriptional activator, a transcriptional repressor, and an expression vector for expressing a nucleic acid encoding a recombinant B4GALT1 gene or B4GALT1 polypeptide. provided. Also provided are therapeutic and prophylactic methods for treating subjects with or at risk of developing cardiovascular conditions.

The wild-type human genomic B4GALT1 nucleic acid is approximately 56.7 kb long, contains six exons, and is located on chromosome 9 in the human genome. An exemplary wild-type human genomic B4GALT1 sequence is assigned to NCBI accession number NG_008919.1 (SEQ ID NO: 1). The variant of human genome B4GALT1 is set forth in SEQ ID NO:2 and contains a single nucleotide polymorphism (SNP) (A to G at position 53576; referred to herein as variant B4GALT1 ). The variant SNP results in a serine at a position corresponding to position 352 in the full-length/mature B4GALT1 polypeptide, rather than the asparagine encoded by the wild-type B4GALT1 polypeptide. As opposed to the three bases "aat" at positions 53575 to 53577 of wild-type human genome B4GALT1 , the variant human genomic B4GALT1 nucleic acid may contain, for example, a serine at a position corresponding to positions 53575 to 53577 of wild-type human genome B4GALT1 . (e.g., “agt”) encoding (compare SEQ ID NO: 2 to SEQ ID NO: 1, respectively). In some embodiments, the isolated nucleic acid molecule comprises SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule consists of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule is the complement of any genomic B4GALT1 nucleic acid molecule disclosed herein.

In some embodiments, the isolated nucleic acid molecule is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, or at least It comprises or consists of nucleic acid sequences that are about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, this nucleic acid sequence also includes nucleotides corresponding to positions 53575 to 53577 of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% of a portion of SEQ ID NO: 2, including exons 1 to 6 of the B4GALT1 gene. , comprises or consists of nucleic acid sequences that are at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, this nucleic acid sequence also includes nucleotides corresponding to positions 53575 to 53577 of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% with a portion of SEQ ID NO:2, including exon 5. , comprises or consists of nucleic acid sequences that are at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, this nucleic acid sequence also includes nucleotides corresponding to positions 53575 to 53577 of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO:2, provided that the nucleic acid sequence includes nucleotides corresponding to positions 53575-53577 of SEQ ID NO:2.

Percent complementarity between specific stretches of nucleic acid sequence within a nucleic acid can be determined using the BLAST program (a basic local alignment search tool) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden , Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, using default settings). It can be determined routinely by using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489), University Research Park, Madison, Wisconsin, USA).

In some embodiments, an isolated nucleic acid molecule comprises less than the entire genome sequence. In some embodiments, the isolated nucleic acid molecule is at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, At least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, at least about 10000, at least about 11000, at least about 12000, at least about 13000, Contains or consists of at least about 14,000, at least about 15,000, at least about 16,000, at least about 17,000, at least about 18,000, at least about 19,000 or at least about 20,000 contiguous nucleotides. In some embodiments, this isolated nucleic acid molecule also includes nucleotides corresponding to positions 53575-53577 of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule is at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900 or at least about Contains or consists of 1000 contiguous nucleotides. In some embodiments, this isolated nucleic acid molecule also includes nucleotides corresponding to positions 53575-53577 of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule has at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least About 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900 or comprises or consists of at least about 1000 contiguous nucleotides. In some embodiments, this isolated nucleic acid molecule also includes nucleotides corresponding to positions 53575-53577 of SEQ ID NO:2.

For example, in some embodiments, the isolated nucleic acid molecule comprises at least 15 contiguous nucleotides of SEQ ID NO:2, wherein the contiguous nucleotides comprise nucleotides 53575 to 53577 of SEQ ID NO:2. In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 contiguous nucleotides of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule comprises 15 to 50 contiguous nucleotides of SEQ ID NO:2, where the contiguous nucleotides comprise nucleotides 53575 to 53577 of SEQ ID NO:2. In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 contiguous nucleotides of SEQ ID NO:2.

In some embodiments, the present disclosure provides an isolated nucleic acid comprising a nucleic acid sequence that is at least 90% identical to a portion of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 comprises nucleotides 53575 to 53577 of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 is at least 15 nucleotides long. In some such embodiments, the portion of SEQ ID NO:2 is at least 20, at least 25, or at least 30 nucleotides long. In some embodiments, the present disclosure provides an isolated nucleic acid comprising a nucleic acid sequence that is at least 90% identical to a portion of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 comprises nucleotides 53575 to 53577 of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 is 15 to 50 nucleotides long. In some such embodiments, the portion of SEQ ID NO:2 is at least 20, at least 25, or at least 30 nucleotides long.

In some embodiments, the present disclosure provides an isolated nucleic acid comprising a nucleic acid sequence that is at least 95% identical to a portion of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 comprises nucleotides 53575 to 53577 of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 is at least 15 nucleotides long. In some such embodiments, the portion of SEQ ID NO:2 is at least 20, at least 25, or at least 30 nucleotides long. In some embodiments, the present disclosure provides an isolated nucleic acid comprising a nucleic acid sequence that is at least 95% identical to a portion of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 comprises nucleotides 53575 to 53577 of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 is 15 to 50 nucleotides long. In some such embodiments, the portion of SEQ ID NO:2 is at least 20, at least 25, or at least 30 nucleotides long.

Such isolated nucleic acid molecules can be used, for example, to express variant B4GALT1 mRNA and protein or as an exogenous donor sequence. It is understood that genetic sequences within a population may vary due to polymorphisms, such as SNPs. The examples provided herein are merely illustrative sequences; other sequences are also possible.

In some embodiments, the isolated nucleic acid molecule comprises a variant B4GALT1 minigene in which one or more non-essential segments of SEQ ID NO:2 are deleted relative to the corresponding wild-type B4GALT1 gene. In some embodiments, the deleted non-essential segment includes one or more intron sequences. In some embodiments, the B4GALT1 minigene may comprise an exon corresponding to any one or more of exons 1 to 6, for example, from variant B4GALT1 (SEQ ID NO: 2), or any combination of such exons. . In some embodiments, the minigene comprises or consists of exon 5 of SEQ ID NO:2. In some embodiments, the B4GALT1 minigene is at least about 70%, at least about 75%, at least about 80% of a portion of SEQ ID NO:2, including any one or more of exons 1 to 6 or any combination of such exons. At least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, the B4GALT1 minigene is at least 70%, at least 75%, at least 80%, at least 85% of a portion of SEQ ID NO:2 comprising any one or more of exons 1 to 6 or any combination of such exons. , is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical, and includes nucleotides corresponding to positions 53575 to 53577 of SEQ ID NO: 2. In some embodiments, the B4GALT1 minigene is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, are at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical.

The present disclosure also provides isolated nucleic acid molecules that hybridize to a variant B4GALT1 genomic sequence or variant B4GALT1 minigene. In some embodiments, such isolated nucleic acid molecules have at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70. , at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least About 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, at least about 10000, at least about 11000, at least about 12000, at least about 13000, at least about 14000 , comprises or consists of at least about 15,000, at least about 16,000, at least about 17,000, at least about 18,000, at least about 19,000 or at least about 20,000 nucleotides. In some embodiments, this isolated nucleic acid molecule also hybridizes to positions 53575-53577 of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule comprises positions 53575 to 53577 of SEQ ID NO:2, or about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40 thereof. , about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides in a segment that hybridizes to a portion of the variant B4GALT1 genome or minigene. In some embodiments, the isolated nucleic acid molecule is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about hybridizes to at least about 15 contiguous nucleotides of the nucleic acid molecule that are 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, this isolated nucleic acid molecule also hybridizes to positions 53575-53577 of SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecule contains or consists of about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides.

For example, in some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising at least 15 nucleotides, wherein the isolated nucleic acid molecule hybridizes to a nucleic acid comprising the sequence of SEQ ID NO: 2, wherein the isolated The nucleic acid molecule hybridizes to a portion of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 includes nucleotides 53575 to 53577 of SEQ ID NO:2. In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 nucleotides. In some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising 15 to 50 nucleotides, wherein the isolated nucleic acid molecule hybridizes to a nucleic acid comprising the sequence of SEQ ID NO: 2, wherein the isolated nucleic acid molecule has Hybridizes to a portion of SEQ ID NO:2, wherein the portion of SEQ ID NO:2 includes nucleotides 53575 to 53577 of SEQ ID NO:2. In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 nucleotides.

In some embodiments, the isolated nucleic acid molecule hybridizes to at least 15 contiguous nucleotides of the nucleic acid, wherein the contiguous nucleotides are at least 90% identical to a portion of SEQ ID NO:2, and wherein the contiguous nucleotides are at positions 53575 to 53577 of SEQ ID NO:2. It includes nucleotides 53575 to 53577 of SEQ ID NO:2 at the corresponding position. In some such embodiments, the contiguous nucleotides are at least 20, at least 25, or at least 30 nucleotides long. In some embodiments, the isolated nucleic acid molecule hybridizes to at least 15 contiguous nucleotides of the nucleic acid, wherein the contiguous nucleotides are at least 95% identical to a portion of SEQ ID NO:2, and wherein the contiguous nucleotides are at positions 53575 to 53577 of SEQ ID NO:2. It includes nucleotides 53575 to 53577 of SEQ ID NO:2 at the corresponding position. In some such embodiments, the contiguous nucleotides are at least 20, at least 25, or at least 30 nucleotides long. In some embodiments, the isolated nucleic acid molecule hybridizes to at least 15 contiguous nucleotides of the nucleic acid, wherein the contiguous nucleotides are at least 100% identical to a portion of SEQ ID NO:2, and wherein the contiguous nucleotides are at positions 53575 to 53577 of SEQ ID NO:2. It includes nucleotides 53575 to 53577 of SEQ ID NO:2 at the corresponding position. In some such embodiments, the contiguous nucleotides are at least 20, at least 25, or at least 30 nucleotides long.

In some embodiments, the isolated nucleic acid molecule hybridizes to 15 to 50 contiguous nucleotides of the nucleic acid, wherein the contiguous nucleotides are at least 90% identical to a portion of SEQ ID NO: 2, and wherein the contiguous nucleotides are positions 53575 to 53577 of SEQ ID NO: 2. It includes nucleotides 53575 to 53577 of SEQ ID NO: 2 at the corresponding position. In some such embodiments, the contiguous nucleotides are at least 20, at least 25, or at least 30 nucleotides long. In some embodiments, the isolated nucleic acid molecule hybridizes to 15 to 50 contiguous nucleotides of the nucleic acid, wherein the contiguous nucleotides are at least 95% identical to a portion of SEQ ID NO: 2, and wherein the contiguous nucleotides are positions 53575 to 53577 of SEQ ID NO: 2. It includes nucleotides 53575 to 53577 of SEQ ID NO: 2 at the corresponding position. In some such embodiments, the contiguous nucleotides are at least 20, at least 25, or at least 30 nucleotides long. In some embodiments, the isolated nucleic acid molecule hybridizes to 15 to 50 contiguous nucleotides of the nucleic acid, wherein the contiguous nucleotides are at least 100% identical to a portion of SEQ ID NO: 2, and wherein the contiguous nucleotides are positions 53575 to 53577 of SEQ ID NO: 2. It includes nucleotides 53575 to 53577 of SEQ ID NO: 2 at the corresponding position. In some such embodiments, the contiguous nucleotides are at least 20, at least 25, or at least 30 nucleotides long.

These isolated nucleic acid molecules can be used, for example, as guide RNAs, primers, probes, or exogenous donor sequences.

A representative wild-type B4GALT1 genome sequence is shown in SEQ ID NO:1. A representative variant B4GALT1 genome sequence variant is shown in SEQ ID NO:2.

The present disclosure also provides isolated nucleic acid molecules comprising variants of B4GALT1 mRNA. The exemplary wild-type human B4GALT1 mRNA is assigned to NCBI accession NM_001497 (SEQ ID NO: 3) and consists of 4214 nucleotide bases. A variant of human B4GALT1 mRNA is set forth in SEQ ID NO:4, which contains a SNP (A to G at position 1244; referred to herein as variant B4GALT1 ), which corresponds to position 352 of the encoded B4GALT1 variant polypeptide. It results in serine at the position. The variant human genomic B4GALT1 mRNA contains, for example, a serine at a position corresponding to positions 1243 to 1245 of the wild-type human B4GALT1 mRNA, as opposed to the three bases "aau" at positions 1243 to 1245 of the wild-type human B4GALT1 mRNA. It contains three bases “agu” encoding (compare SEQ ID NO: 4 with SEQ ID NO: 3, respectively). In some embodiments, the isolated nucleic acid molecule comprises SEQ ID NO:4. In some embodiments, the isolated nucleic acid molecule consists of SEQ ID NO:4.

In some embodiments, the isolated nucleic acid molecule is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least It comprises or consists of nucleic acid sequences that are about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, this nucleic acid sequence also includes nucleotides corresponding to positions 1243-1245 of SEQ ID NO:4. In some embodiments, the isolated nucleic acid molecule is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about a portion of SEQ ID NO:4, including exons 1-6. It comprises or consists of nucleotide sequences that are 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, this nucleic acid sequence also includes nucleotides corresponding to positions 1243-1245 of SEQ ID NO:4. In some embodiments, the isolated nucleic acid molecule is the complement of any B4GALT1 mRNA molecule disclosed herein.

In some embodiments, the isolated nucleic acid molecule comprises less than the entire mRNA sequence. In some embodiments, the isolated nucleic acid molecule is at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, At least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about Contains or consists of 1000, at least about 2000, at least about 3000 or at least about 4000 contiguous nucleotides. In some embodiments, this isolated nucleic acid molecule also includes nucleotides corresponding to positions 1243-1245 of SEQ ID NO:4. In some embodiments, the isolated nucleic acid molecule is at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900 or at least about Contains or consists of 1000 contiguous nucleotides. In some embodiments, this isolated nucleic acid molecule also includes nucleotides corresponding to positions 1243-1245 of SEQ ID NO:4. In some embodiments, the isolated nucleic acid molecule has at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50 of exons 1 to 6 of SEQ ID NO:4. , at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least Contains or consists of about 900 or at least about 1000 contiguous nucleotides. In some embodiments, this isolated nucleic acid molecule also includes nucleotides corresponding to positions 1243-1245 of SEQ ID NO:4.

In some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence that is at least 90% identical to a portion of SEQ ID NO:4, wherein the portion of SEQ ID NO:4 comprises nucleotides 1243 to 1245 of SEQ ID NO:4 , wherein the portion of SEQ ID NO:4 includes at least 15 nucleotides of SEQ ID NO:4. In some such embodiments, the portion of SEQ ID NO:4 is at least 20, at least 25, or at least 30 nucleotides of SEQ ID NO:4. In some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence that is at least 95% identical to a portion of SEQ ID NO:4, wherein the portion of SEQ ID NO:4 comprises nucleotides 1243 to 1245 of SEQ ID NO:4 , wherein the portion of SEQ ID NO:4 includes at least 15 nucleotides of SEQ ID NO:4. In some such embodiments, the portion of SEQ ID NO:4 is at least 20, at least 25, or at least 30 nucleotides of SEQ ID NO:4. In some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence that is at least 100% identical to a portion of SEQ ID NO:4, wherein the portion of SEQ ID NO:4 comprises nucleotides 1243 to 1245 of SEQ ID NO:4 , wherein the portion of SEQ ID NO:4 includes at least 15 nucleotides of SEQ ID NO:4. In some such embodiments, the portion of SEQ ID NO:4 is at least 20, at least 25, or at least 30 nucleotides of SEQ ID NO:4. In some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence that is at least 90% identical to a portion of SEQ ID NO:4, wherein the portion of SEQ ID NO:4 comprises nucleotides 1243 to 1245 of SEQ ID NO:4 , wherein the portion of SEQ ID NO: 4 includes 15 to 50 nucleotides of SEQ ID NO: 4. In some such embodiments, the portion of SEQ ID NO:4 is at least 20, at least 25, or at least 30 nucleotides of SEQ ID NO:4. In some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence that is at least 95% identical to a portion of SEQ ID NO:4, wherein the portion of SEQ ID NO:4 comprises nucleotides 1243 to 1245 of SEQ ID NO:4 , wherein the portion of SEQ ID NO:4 includes 15 to 50 nucleotides of SEQ ID NO:4. In some such embodiments, the portion of SEQ ID NO:4 is at least 20, at least 25, or at least 30 nucleotides of SEQ ID NO:4. In some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence that is 100% identical to a portion of SEQ ID NO:4, wherein the portion of SEQ ID NO:4 comprises nucleotides 1243 to 1245 of SEQ ID NO:4, Here, the portion of SEQ ID NO: 4 includes 15 to 50 nucleotides of SEQ ID NO: 4. In some such embodiments, the portion of SEQ ID NO:4 is at least 20, at least 25, or at least 30 nucleotides of SEQ ID NO:4.

Such isolated nucleic acid molecules can be used, for example, to express a B4GALT1 variant polypeptide or as an exogenous donor sequence. It is understood that genetic sequences within a population may vary due to polymorphisms, such as SNPs. The examples provided herein are merely illustrative sequences; other sequences are also possible.

In some embodiments, the isolated nucleic acid molecule is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92% with the variant Asn352Ser B4GALT1 polypeptide (SEQ ID NO: 8). , or comprises a nucleic acid sequence encoding a polypeptide that is at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. It consists of, provided that the polypeptide contains serine at the position corresponding to position 352. In some embodiments, the isolated nucleic acid molecule comprises or consists of a nucleic acid sequence encoding a polypeptide that is at least about 90% identical to SEQ ID NO: 8, provided that the polypeptide contains a serine at a position corresponding to position 352. . In some embodiments, the isolated nucleic acid molecule comprises or consists of a nucleic acid sequence encoding a polypeptide that is at least about 95% identical to SEQ ID NO: 8, provided that the polypeptide contains a serine at a position corresponding to position 352. .

For example, in some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence that is at least 10 amino acids in length, wherein the amino acid sequence is a portion of the amino acid sequence of SEQ ID NO: 8 and 90% of the amino acid sequence. identical, wherein some contain serine at a position corresponding to position 352 in SEQ ID NO:8. In some such embodiments, the nucleic acid sequence encodes a polypeptide having an amino acid sequence that is at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence that is at least 10 amino acids in length, wherein the amino acid sequence is 95% identical to a portion of the amino acid sequence of SEQ ID NO: 8, wherein Some contain serine at a position corresponding to position 352 in SEQ ID NO:8. In some such embodiments, the nucleic acid sequence encodes a polypeptide having an amino acid sequence that is at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence that is 10 to 50 amino acids in length, wherein the amino acid sequence is 90% identical to a portion of the amino acid sequence of SEQ ID NO: 8, Here, some contain serine at a position corresponding to position 352 in SEQ ID NO:8. In some such embodiments, the nucleic acid sequence encodes a polypeptide having an amino acid sequence that is at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence that is 10 to 50 amino acids in length, wherein the amino acid sequence is 95% identical to a portion of the amino acid sequence of SEQ ID NO: 8, Here, some contain serine at a position corresponding to position 352 in SEQ ID NO:8. In some such embodiments, the nucleic acid sequence encodes a polypeptide having an amino acid sequence that is at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the isolated nucleic acid molecule comprises or consists of a nucleic acid sequence encoding a polypeptide identical to SEQ ID NO:8.

The present disclosure also provides isolated nucleic acid molecules that hybridize to variant B4GALT1 mRNA sequences. In some embodiments, such isolated nucleic acid molecules have at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70. , at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least Contains or consists of about 2000, at least about 3000 or at least about 4000 nucleotides. In some embodiments, this isolated nucleic acid molecule also hybridizes to positions 1243-1245 of SEQ ID NO:4. In some embodiments, the isolated nucleic acid molecule comprises positions 1243-1245 of SEQ ID NO:4, or about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40 thereof. , hybridizes to a portion of the variant B4GALT1 mRNA in a segment within about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides.

In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and contains a variant B4GALT1 mRNA (e.g. For example, it hybridizes to part of SEQ ID NO: 4). In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and contains a variant B4GALT1 mRNA (e.g. For example, it hybridizes to a portion of SEQ ID NO: 4) and hybridizes to positions 1243 to 1245 of SEQ ID NO: 4. In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises 15 to 50 nucleotides and hybridizes to a portion of the variant B4GALT1 mRNA (e.g., SEQ ID NO: 4) in a segment comprising positions 1243 to 1245 of SEQ ID NO: 4. , hybridizes to positions 1243 to 1245 of SEQ ID NO: 4. In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 nucleotides.

In some embodiments, the isolated nucleic acid molecule is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, the isolated nucleic acid molecule also hybridizes to positions 1243-1245 of SEQ ID NO:4. In some embodiments, the isolated nucleic acid molecule contains or consists of about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides.

In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is located in a portion of the variant B4GALT1 mRNA in a segment comprising or within 5 nucleotides of positions 1243 to 1245 of SEQ ID NO:4. hybridized, wherein the variant B4GALT1 mRNA is at least 90% identical to the variant B4GALT1 mRNA (e.g., SEQ ID NO:4). In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is located in a portion of the variant B4GALT1 mRNA in a segment comprising or within 5 nucleotides of positions 1243 to 1245 of SEQ ID NO:4. hybridized, wherein the variant B4GALT1 mRNA is at least 95% identical to the variant B4GALT1 mRNA (e.g., SEQ ID NO:4). In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is located in a portion of the variant B4GALT1 mRNA in a segment comprising or within 5 nucleotides of positions 1243 to 1245 of SEQ ID NO:4. hybridizes to positions 1243 to 1245 of SEQ ID NO:4, wherein the variant B4GALT1 mRNA is at least 90% identical to the variant B4GALT1 mRNA (e.g., SEQ ID NO:4). In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is located in a portion of the variant B4GALT1 mRNA in a segment comprising or within 5 nucleotides of positions 1243 to 1245 of SEQ ID NO:4. hybridizes to positions 1243 to 1245 of SEQ ID NO:4, wherein the variant B4GALT1 mRNA is at least 95% identical to the variant B4GALT1 mRNA (e.g., SEQ ID NO:4). In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 nucleotides. In some embodiments, the isolated nucleic acid molecule contains or consists of 15 to 100 nucleotides, or 15 to 35 nucleotides.

These isolated nucleic acid molecules can be used, for example, as guide RNAs, primers, probes, or exogenous donor sequences.

A representative wild-type B4GALT1 mRNA sequence is shown in SEQ ID NO:3. A representative variant B4GALT1 mRNA sequence is shown in SEQ ID NO:4.

The present disclosure also provides nucleic acid molecules comprising variants of B4GALT1 cDNA encoding all or part of a B4GALT1 variant polypeptide. An exemplary wild-type human B4GALT1 cDNA (e.g., the coding region of mRNA, written DNA) consists of 1197 nucleotide bases (SEQ ID NO: 5). A variant of human B4GALT1 cDNA is set forth in SEQ ID NO:6, which contains a SNP (A to G at position 1055; referred to herein as variant B4GALT1 ), which corresponds to position 352 of the encoded B4GALT1 variant polypeptide. It results in serine at the position. As opposed to the three bases "aat" of the wild-type human B4GALT1 cDNA at positions 1054 to 1056, the variant human genomic B4GALT1 cDNA has, for example, positions corresponding to positions 1054 to 1056 of the full-length/mature wild-type human B4GALT1 cDNA. and "agt", which encodes serine (compare SEQ ID NO: 6 with SEQ ID NO: 5, respectively). In some embodiments, the nucleic acid molecule comprises SEQ ID NO:6. In some embodiments, the nucleic acid molecule consists of SEQ ID NO:6. In some embodiments, the cDNA molecule is isolated.

In some embodiments, the cDNA molecule is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% %, at least about 98%, at least about 99%, or 100% identical nucleic acid sequences. In some embodiments, the cDNA molecule also includes nucleotides corresponding to positions 1054-1056 of SEQ ID NO:6. In some embodiments, the isolated nucleic acid molecule is the complement of any B4GALT1 cDNA molecule disclosed herein.

In some embodiments, the cDNA molecule includes less than the entire cDNA sequence. In some embodiments, the cDNA molecule has at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, or Contains or consists of at least about 1100 contiguous nucleotides. In some embodiments, this cDNA molecule also includes nucleotides corresponding to positions 1054-1056 of SEQ ID NO:6. In some embodiments, the cDNA molecule has at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about Contains or consists of 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400 or at least about 500 contiguous nucleotides. In some embodiments, this cDNA molecule also includes nucleotides corresponding to positions 1054-1056 of SEQ ID NO:6.

For example, in some embodiments, the cDNA molecule comprises at least 15 contiguous nucleotides of SEQ ID NO:6, wherein the contiguous nucleotides comprise nucleotides 1054-1056 of SEQ ID NO:6. In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 contiguous nucleotides of SEQ ID NO:6. In some embodiments, the cDNA molecule comprises 15 to 50 contiguous nucleotides of SEQ ID NO:6, where the contiguous nucleotides comprise nucleotides 1054 to 1056 of SEQ ID NO:6. In some such embodiments, the isolated nucleic acid molecule comprises at least 20, at least 25, or at least 30 contiguous nucleotides of SEQ ID NO:6. In some embodiments, the present disclosure provides a cDNA molecule comprising a nucleic acid sequence that is at least 90% identical to a portion of SEQ ID NO:6, wherein the portion of SEQ ID NO:6 comprises nucleotides 1054 to 1056 of SEQ ID NO:6, wherein A portion of SEQ ID NO:6 includes at least 15 consecutive nucleotides of SEQ ID NO:6. In some such embodiments, the portion of SEQ ID NO:6 is at least 20, at least 25, or at least 30 consecutive nucleotides of SEQ ID NO:6. In some embodiments, the present disclosure provides a cDNA molecule comprising a nucleic acid sequence that is at least 95% identical to a portion of SEQ ID NO:6, wherein the portion of SEQ ID NO:6 comprises nucleotides 1054 to 1056 of SEQ ID NO:6, wherein A portion of SEQ ID NO:6 includes at least 15 consecutive nucleotides of SEQ ID NO:6. In some such embodiments, the portion of SEQ ID NO:6 is at least 20, at least 25, or at least 30 consecutive nucleotides of SEQ ID NO:6. In some embodiments, the present disclosure provides a cDNA molecule comprising a nucleic acid sequence that is at least 90% identical to a portion of SEQ ID NO:6, wherein the portion of SEQ ID NO:6 comprises nucleotides 1054 to 1056 of SEQ ID NO:6, wherein A portion of SEQ ID NO:6 includes 15 to 50 consecutive nucleotides of SEQ ID NO:6. In some such embodiments, the portion of SEQ ID NO:6 is at least 20, at least 25, or at least 30 consecutive nucleotides of SEQ ID NO:6. In some embodiments, the present disclosure provides a cDNA molecule comprising a nucleic acid sequence that is at least 95% identical to a portion of SEQ ID NO:6, wherein the portion of SEQ ID NO:6 comprises nucleotides 1054 to 1056 of SEQ ID NO:6, wherein A portion of SEQ ID NO:6 includes 15 to 50 consecutive nucleotides of SEQ ID NO:6. In some such embodiments, the portion of SEQ ID NO:6 is at least 20, at least 25, or at least 30 consecutive nucleotides of SEQ ID NO:6. In some embodiments, the present disclosure provides a cDNA molecule comprising nucleotides 1054 to 1056 of SEQ ID NO:6 at a position corresponding to nucleotides 1054 to 1056 of SEQ ID NO:6, wherein the cDNA molecule comprises a portion of SEQ ID NO:6 and at least and wherein the portion of SEQ ID NO:6 comprises nucleotides 1054 to 1056 of SEQ ID NO:6, and wherein the portion of SEQ ID NO:6 comprises at least 15 consecutive nucleotides of SEQ ID NO:6. In some such embodiments, the portion of SEQ ID NO:6 is at least 20, at least 25, or at least 30 consecutive nucleotides of SEQ ID NO:6. In some embodiments, the present disclosure provides a cDNA molecule comprising nucleotides 1054 to 1056 of SEQ ID NO:6 at a position corresponding to nucleotides 1054 to 1056 of SEQ ID NO:6, wherein the cDNA molecule comprises a portion of SEQ ID NO:6 and at least and wherein the portion of SEQ ID NO:6 comprises nucleotides 1054 to 1056 of SEQ ID NO:6, and wherein the portion of SEQ ID NO:6 comprises at least 15 consecutive nucleotides of SEQ ID NO:6. In some such embodiments, the portion of SEQ ID NO:6 is at least 20, at least 25, or at least 30 consecutive nucleotides of SEQ ID NO:6. In some embodiments, the present disclosure provides a cDNA molecule comprising nucleotides 1054 to 1056 of SEQ ID NO:6 at a position corresponding to nucleotides 1054 to 1056 of SEQ ID NO:6, wherein the cDNA molecule comprises a portion of SEQ ID NO:6 and at least and wherein the portion of SEQ ID NO:6 comprises nucleotides 1054 to 1056 of SEQ ID NO:6, and wherein the portion of SEQ ID NO:6 comprises 15 to 50 contiguous nucleotides of SEQ ID NO:6. In some such embodiments, the portion of SEQ ID NO:6 is at least 20, at least 25, or at least 30 consecutive nucleotides of SEQ ID NO:6. In some embodiments, the present disclosure provides a cDNA molecule comprising nucleotides 1054 to 1056 of SEQ ID NO:6 at a position corresponding to nucleotides 1054 to 1056 of SEQ ID NO:6, wherein the cDNA molecule comprises a portion of SEQ ID NO:6 and at least and wherein the portion of SEQ ID NO:6 comprises nucleotides 1054 to 1056 of SEQ ID NO:6, and wherein the portion of SEQ ID NO:6 comprises 15 to 50 contiguous nucleotides of SEQ ID NO:6. In some such embodiments, the portion of SEQ ID NO:6 is at least 20, at least 25, or at least 30 consecutive nucleotides of SEQ ID NO:6.

Such cDNA molecules can be used, for example, to express a B4GALT1 variant protein or as an exogenous donor sequence. It is understood that genetic sequences within a population may vary due to polymorphisms, such as SNPs. The examples provided herein are merely illustrative sequences; other sequences are also possible.

In some embodiments, the cDNA molecule is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least comprising or consisting of a nucleic acid sequence encoding a polypeptide that is about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical; However, the polypeptide contains serine at the position corresponding to position 352. In some embodiments, the cDNA molecule comprises or consists of a nucleic acid sequence encoding a polypeptide that is at least about 90% identical to SEQ ID NO:8, provided that the polypeptide includes a serine at a position corresponding to position 352. In some embodiments, the cDNA molecule comprises or consists of a nucleic acid sequence encoding a polypeptide that is at least about 95% identical to SEQ ID NO:8, provided that the polypeptide includes a serine at a position corresponding to position 352. In some embodiments, the cDNA molecule comprises or consists of a nucleic acid sequence encoding a polypeptide identical to SEQ ID NO:8.

The present disclosure also provides isolated nucleic acid molecules that hybridize to variant B4GALT1 cDNA sequences. In some embodiments, such isolated nucleic acid molecules have at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70. , at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, or Contains or consists of at least about 1100 nucleotides. In some embodiments, this isolated nucleic acid molecule also hybridizes to positions 1054-1056 of SEQ ID NO:6. In some embodiments, such isolated nucleic acid molecule comprises positions 1054-1056 of SEQ ID NO:6 or about 600, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about hybridizes to a portion of the variant B4GALT1 cDNA at a segment within 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides. In some embodiments, the isolated nucleic acid molecule is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about hybridizes to at least about 15 contiguous nucleotides of the cDNA molecule that are 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, the isolated nucleic acid molecule also hybridizes to positions 1054-1056 of SEQ ID NO:6. In some embodiments, the isolated nucleic acid molecule contains or consists of about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides.

In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is attached to a portion of the variant B4GALT1 cDNA in a segment comprising or within 5 nucleotides of positions 1054 to 1056 of SEQ ID NO:6. hybridized, wherein the variant B4GALT1 cDNA is at least 90% identical to the variant B4GALT1 cDNA (e.g., SEQ ID NO:6). In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is attached to a portion of the variant B4GALT1 cDNA in a segment comprising or within 5 nucleotides of positions 1054 to 1056 of SEQ ID NO:6. hybridized, wherein the variant B4GALT1 cDNA is at least 95% identical to the variant B4GALT1 cDNA (e.g., SEQ ID NO:6). In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is attached to a portion of the variant B4GALT1 cDNA in a segment comprising or within 5 nucleotides of positions 1054 to 1056 of SEQ ID NO:6. hybridized, wherein the variant B4GALT1 cDNA is 100% identical to the variant B4GALT1 cDNA (e.g., SEQ ID NO:6). In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is attached to a portion of the variant B4GALT1 cDNA in a segment comprising or within 5 nucleotides of positions 1054 to 1056 of SEQ ID NO:6. Hybridizes to positions 1054 to 1056 of SEQ ID NO:6, wherein the variant B4GALT1 cDNA is at least 90% identical to the variant B4GALT1 cDNA (e.g., SEQ ID NO:6). In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is attached to a portion of the variant B4GALT1 cDNA in a segment comprising or within 5 nucleotides of positions 1054 to 1056 of SEQ ID NO:6. hybridizes to positions 1054 to 1056 of SEQ ID NO:6, wherein the variant B4GALT1 cDNA is at least 95% identical to the variant B4GALT1 cDNA (e.g., SEQ ID NO:6). In some embodiments, the isolated nucleic acid molecule comprises or consists of at least 15 nucleotides and is attached to a portion of the variant B4GALT1 cDNA in a segment comprising or within 5 nucleotides of positions 1054 to 1056 of SEQ ID NO:6. hybridized to positions 1054 to 1056 of SEQ ID NO:6, wherein the variant B4GALT1 cDNA is 100% identical to the variant B4GALT1 cDNA (e.g., SEQ ID NO:6). In some embodiments, the isolated nucleic acid molecule contains or consists of 15 to 100 nucleotides, or 15 to 35 nucleotides.

These isolated nucleic acid molecules can be used, for example, as guide RNAs, primers, probes, exogenous donor sequences, antisense RNA, siRNA or shRNA.

A representative wild-type B4GALT1 cDNA sequence is shown in SEQ ID NO:5. A representative variant B4GALT1 cDNA sequence is shown in SEQ ID NO:6.

Nucleic acid molecules disclosed herein may comprise the nucleic acid sequence of a naturally occurring B4GALT1 gene or mRNA transcript, or may comprise non-naturally occurring sequences. In some embodiments, a naturally occurring sequence may differ from a non-naturally occurring sequence due to synonymous mutations or mutations that do not affect the encoded B4GALT1 polypeptide. For example, the sequences may be identical except for synonymous mutations or mutations that do not affect the encoded B4GALT1 polypeptide. A synonymous mutation or substitution is the substitution of one nucleotide for another nucleotide within an exon of a gene encoding a protein such that the resulting amino acid sequence is not altered. This is possible due to the degenerate nature of the genetic code, where some amino acids are encoded by more than one 3-base pair codon. Synonymous substitutions are used, for example, in the process of codon optimization. Nucleic acid molecules disclosed herein can be codon optimized.

Also provided herein are functional polynucleotides capable of interacting with the disclosed nucleic acid molecules. A functional polynucleotide is a nucleic acid molecule that has a specific function, such as binding to a target molecule or catalyzing a specific reaction. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences. Functional polynucleotides can act as effectors, inhibitors, modulators and stimulators of the specific activity possessed by the target molecule, or functional polynucleotides can possess de novo activity independently of any other molecule.

Antisense molecules are designed to interact with target nucleic acid molecules through canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote destruction of the target molecule, for example, through RNase-H-mediated RNA-DNA hybrid cleavage. Alternatively, antisense molecules are designed to interfere with processing functions that would normally occur on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Various methods exist for optimization of antisense efficiency by identifying the most accessible region of the target molecule. Examples include, but are not limited to, in vitro selection experiments, and DNA modification studies using DMS and DEPC. Antisense molecules generally bind target molecules with a dissociation constant (k _d ) of about 10 ^-6 or less, about 10 ^-8 or less, about 10 ^{-10 or} less, or about 10 ^{-12 or} less. Representative samples of methods and techniques assisting in the design and use of antisense molecules can be found in the following non-limiting list of U.S. patents: U.S. Patent 5,135,917; 5,294,533; 5,627,158; 5,641,754; 5,691,317; 5,780,607; 5,786,138; 5,849,903; 5,856,103; 5,919,772; 5,955,590; 5,990,088; 5,994,320; 5,998,602; 6,005,095; 6,007,995; 6,013,522; 6,017,898; 6,018,042; 6,025,198; 6,033,910; 6,040,296; 6,046,004; 6,046,319; and 6,057,437. Examples of antisense molecules include, but are not limited to, antisense RNA, short interfering RNA (siRNA), and short hairpin RNA (shRNA).

Isolated nucleic acid molecules disclosed herein may include RNA, DNA, or both RNA and DNA. Isolated nucleic acid molecules can also be linked or fused to heterologous nucleic acid sequences, for example, in vectors, or heterologous labels. For example, an isolated nucleic acid molecule disclosed herein can be presented within a vector or exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. Isolated nucleic acid molecules can also be linked or fused to heterologous labels, such as fluorescent labels. Other examples of labels are disclosed elsewhere herein.

The label may be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, enzyme, or fluorophore quencher). Such labels may be detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Such labels include, for example, radioactive labels that can be measured with a radiation-counting device; Pigments, dyes or other color sources that can be observed visually or measured spectrophotometrically; Spin label, which can be measured with a spin label analyzer; and a fluorescent label (e.g., a fluorophore), wherein the output signal is produced by excitation of a suitable molecular adduct and can be visualized by excitation with light absorbed by the dye, or by standard fluorometer or It can be measured with an imaging system. Labels may also include, for example, chemiluminescent materials (where the output signal is produced by chemical modification of the signal compound); metal-containing materials; or an enzyme, where enzyme-dependent secondary production of a signal occurs, e.g., formation of a colored product from a colorless substrate. The term “label” can also refer to a “tag” or hapten that can selectively bind to a conjugated molecule such that it is used to produce a detectable signal when the conjugated molecule is later added with a substrate. For example, biotin can be used as a tag, followed by binding to the tag using an avidin or streptavidin conjugate of horseradish peroxidate (HRP), followed by a calorimetric substrate (e.g., tetramethyl benzidine ( TMB)) or fluorogenic substrates can be used to detect the presence of HRP. Exemplary labels include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Many labels are known and include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent and chemiluminescent substrates and other labels.

The disclosed nucleic acid molecules may consist, for example, of nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutions. Such nucleotides include nucleotides that contain modified bases, sugar or phosphate groups, or incorporate non-natural moieties into the structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.

Nucleic acid molecules disclosed herein may also include one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides that contain modifications to a base, sugar, or phosphate moiety. Modifications to base moieties include natural and synthetic modifications of A, C, G and T/U, as well as different purine or pyrimidine bases, such as pseudouridine, uracil-5-yl, hypoxanthine-9-yl ( I) and 2-aminoadenin-9-yl. The modified bases are 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, adenine and guanine. 2-propyl and other alkyl derivatives of, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azouracil, cytosine and Thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, In particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaguanine. -Includes, but is not limited to, deazadenine and 3-deazaguanine and 3-deazadenine. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines such as, but not limited to, 2-aminopropyladenine, 5-pro Pinyluracil, 5-propynylcytosine and 5-methylcytosine can increase the stability of duplex formation. Usually, base modifications can be combined, for example, with sugar modifications, such as 2'-O-methoxyethyl, to achieve unique properties, such as increased duplex stability.

Nucleotide analogs may also include modifications of sugar moieties. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl can be substituted or unsubstituted C _1-10 alkyl or C _2-10 alkenyl and C _2-10 alkynyl. Exemplary 2' sugar modifications also include -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , -O(CH ₂ ) _n -ONH ₂ , and -O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ (wherein n and m are 1 to about 10); It is not limited to these.

Other modifications at the 2' position include C _1-10 alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cutter , reporter groups, intercalators, groups for improving the pharmacokinetic properties of an oligonucleotide or groups for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, especially on the 3' terminal nucleotide or at the 3' position of the sugar in a 2'-5' linked oligonucleotide and the 5' position of the 5' terminal nucleotide. Modified sugars may also include those containing modifications at bridging ring oxygens, such as CH ₂ and S. Nucleotide sugar analogs can also have a sugar mimetic, such as a cyclobutyl moiety, in place of the pentofuranosyl sugar.

Nucleotide analogs may also be modified at the phosphate moiety. Modified phosphate moieties are those in which the linkage between two nucleotides is phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphothryester, aminoalkylphosphothryester, 3'-alkylene phosphonate, and phosphorothioate. Methyl and other alkyl phosphonates, including iral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, ti Including, but not limited to, those that can be modified to contain onoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. This phosphate or modified phosphate linkage between two nucleotides may be through a 3'-5' linkage or a 2'-5' linkage, with the linkage having reversed polarity, e.g., 3'-5' to 5'-3'. Or it may contain 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

Nucleotide substituents include molecules that have functional properties similar to nucleotides but do not contain a phosphate moiety, such as peptide nucleic acids (PNAs). Nucleotide substituents will recognize nucleic acids in Watson-Crick or Hoogsteen fashion, but include molecules that are linked together through moieties other than the phosphate moiety. Nucleotide substituents can align to a double helix type structure when interacting with an appropriate target nucleic acid.

Nucleotide substituents also include nucleotides or nucleotide analogs in which a phosphate moiety or sugar moiety has been replaced. In some embodiments, the nucleotide substituent cannot contain a standard phosphorus atom. Substituents for phosphate include, for example, linkages between short chain alkyl or cycloalkyl nucleosides, linkages between mixed heteroatoms and alkyl or cycloalkyl nucleosides, or one or more short chain heteroatoms or heterocyclic nucleosides. It may be a liver linkage. These include morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane skeleton; sulfide, sulfoxide and sulfone skeletons; Formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; Alkene-containing skeleton; Sulfamate skeleton; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide backbones; amide skeleton; and others having mixed N, O, S and CH ₂ component portions.

It is also understood that in nucleotide substitutions, both the sugar and phosphate moieties of the nucleotide can be replaced, for example by an amide type linkage (aminoethylglycine) (PNA).

It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs, for example to improve cellular uptake. Conjugates may be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, for example, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl moieties, phos Polylipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, adamantane acetic acid, palmityl moiety or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

The disclosure also provides vectors comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules and heterologous nucleic acids disclosed herein. Vectors can be viral or non-viral vectors capable of transporting nucleic acid molecules. In some embodiments, the vector is a plasmid or cosmid (e.g., circular double-stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector, where additional DNA segments can be ligated within the viral genome. In some embodiments, the vector is capable of replicating autonomously in the host cell into which it is introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). In some embodiments, a vector (e.g., a non-episomal mammalian vector) can integrate into the genome of a host cell upon introduction into the host cell, thereby replicating with the host genome. Additionally, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” or “expression vectors”. Such vectors may also be targeting vectors (i.e., exogenous donor sequences).

In some embodiments, the proteins encoded by the various genetic variants disclosed herein can be derived from nucleic acid molecules encoding the disclosed genetic variants such that these genes are operably linked to expression control sequences, such as transcription and translation control sequences, using expression vectors. It is expressed by inserting it into the body. Expression vectors include plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosome (YAC), Epstein-Barr (EBV)- Including, but not limited to, derived episomes, etc. In some embodiments, a nucleic acid molecule comprising a disclosed genetic variant can be ligated into a vector such that transcriptional and translational control sequences within the vector provide its intended function of regulating transcription and translation of the genetic variant. Expression vectors and expression control sequences are selected to be compatible with the expression host cells used. Nucleic acid sequences comprising the disclosed genetic variants can be inserted into separate vectors or into the same expression vector as the variant genetic information. Nucleic acid sequences comprising the disclosed genetic variants can be inserted into expression vectors by standard methods (e.g., ligation of complementary restriction sites on the nucleic acid containing the disclosed genetic variants and the vector, or blunt end ligation if restriction sites are not present). You can.

In addition to the nucleic acid sequence containing the disclosed genetic variant, the recombinant expression vector may contain regulatory sequences that control expression of the genetic variant in the host cell. The design of the expression vector, including the choice of control sequences, may depend on factors such as the choice of host cell to be transformed, the expression level of the protein of interest, etc. Regulatory sequences of interest for mammalian host cell expression include, for example, viral elements that direct high level protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter/enhancer) ), simian virus 40 (SV40) (e.g. SV40 promoter/enhancer), promoters and/or enhancers derived from adenoviruses (e.g. adenovirus major late promoter (AdMLP)), polyomas and strong mammalian promoters, For example, it may include native immunoglobulin and actin promoter. Methods for expressing polypeptides in bacterial cells or fungal cells (e.g., yeast cells) are also well known.

A promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific promoter). or tissue-specific promoter). Examples of promoters can be found, for example, in International Patent No. WO 2013/176772.

Examples of inducible promoters include, for example, chemically controlled promoters and physically controlled promoters. Chemically controlled promoters include, for example, alcohol-controlled promoters (e.g., alcohol dehydrogenase (alcA) gene promoter), tetracycline-controlled promoters (e.g., tetracycline-responsive promoters, tetracycline operator sequences (tetO), tet-on promoter or tet-off promoter), a steroid-regulated promoter (e.g., the rat glucocorticoid receptor, the promoter of the estrogen receptor, or the promoter of the ecdysone receptor), or a metal-regulated promoter (e.g., the metal low protein promoter). Physically regulated promoters include, for example, temperature-regulated promoters (e.g., heat shock promoters) and light-regulated promoters (e.g., light-inducible promoters or light-repressible promoters).

Tissue-specific promoters include, for example, neuron-specific promoters, glial cell-specific promoters, muscle cell-specific promoters, cardiac cell-specific promoters, kidney cell-specific promoters, bone cell-specific promoters, It may be an endothelial cell-specific promoter, or an immune cell-specific promoter (e.g., a B cell promoter or a T cell promoter).

Developmentally regulated promoters include, for example, promoters that are active only during the embryonic phase of development, or only in adult cells.

In addition to the nucleic acid sequences comprising the disclosed genetic variants and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. there is. Selectable marker genes can facilitate the selection of host cells into which the vector has been introduced (see, for example, US Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, a selectable marker gene can confer resistance to a drug, such as G418, hygromycin, or methotrexate, in the host cell into which the vector has been introduced. Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification), the neo gene (for G418 selection), and glutamate synthase (GS ) genes, but are not limited to these.

The present disclosure also provides isolated polypeptides comprising variant B4GALT1 polypeptides (Asn352Ser). The exemplary wild-type human B4GALT1 polypeptide is assigned UniProt accession number P15291 (SEQ ID NO: 7) and consists of 398 amino acids. In contrast to the asparagine at the same position in wild-type human B4GALT1 , the human variant B4GALT1 polypeptide contains a serine at the position corresponding to position 352 of the full-length/mature B4GALT1 polypeptide (SEQ ID NO: 8) (respectively SEQ ID NO: 8) Compare with number 7). In some embodiments, the isolated polypeptide comprises SEQ ID NO:8. In some embodiments, the isolated polypeptide consists of SEQ ID NO:8.

In some embodiments, the isolated polypeptide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, or at least It comprises or consists of amino acid sequences that are about 97%, at least about 98%, at least about 99%, or 100% identical. In some embodiments, the isolated polypeptide comprises a serine at a position corresponding to position 352 of SEQ ID NO:8. In some embodiments, the isolated polypeptide has an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:8. Contains or consists of this. In some embodiments, the isolated polypeptide comprises a serine at a position corresponding to position 352 of SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 90% identical to SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 90% identical to SEQ ID NO: 8 and includes a serine at a position corresponding to position 352 of SEQ ID NO: 8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 90% identical to SEQ ID NO: 8, provided that the isolated polypeptide contains a serine at a position corresponding to position 352 of SEQ ID NO: 8. do.

In some embodiments, the isolated polypeptide comprises a serine at a position corresponding to position 352 of SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 95% identical to SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 95% identical to SEQ ID NO: 8 and includes a serine at a position corresponding to position 352 of SEQ ID NO: 8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 95% identical to SEQ ID NO: 8, provided that the isolated polypeptide contains a serine at a position corresponding to position 352 of SEQ ID NO: 8. do. In some embodiments, the isolated polypeptide comprises a serine at a position corresponding to position 352 of SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 98% identical to SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 98% identical to SEQ ID NO: 8 and includes a serine at a position corresponding to position 352 of SEQ ID NO: 8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 98% identical to SEQ ID NO: 8, provided that the isolated polypeptide contains a serine at a position corresponding to position 352 of SEQ ID NO: 8. do. In some embodiments, the isolated polypeptide comprises a serine at a position corresponding to position 352 of SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 99% identical to SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 99% identical to SEQ ID NO: 8 and includes a serine at a position corresponding to position 352 of SEQ ID NO: 8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least about 99% identical to SEQ ID NO: 8, provided that the isolated polypeptide contains a serine at a position corresponding to position 352 of SEQ ID NO: 8. do.

In some embodiments, the isolated polypeptide is at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, It contains or consists of at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300 or at least about 350 contiguous amino acids. In some embodiments, the isolated polypeptide also includes a serine at a position corresponding to position 352 of SEQ ID NO:8. In some embodiments, the isolated polypeptide is at least about 8, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300 or at least about 350 contiguous amino acids and at least about 70 %, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96 %, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences. In some embodiments, the isolated polypeptide also includes a serine at a position corresponding to position 352 of SEQ ID NO:8. In some embodiments, the isolated polypeptide is at least about 8, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300 or at least about 350 contiguous amino acids and at least about 90 %, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical Contains or consists of an amino acid sequence. In some embodiments, the isolated polypeptide also includes a serine at a position corresponding to position 352 of SEQ ID NO:8.

In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least 90% identical to at least 300 contiguous amino acids of SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least 90% identical to at least 300 contiguous amino acids of SEQ ID NO: 8, and the isolated polypeptide also has an amino acid sequence corresponding to position 352 of SEQ ID NO: 8. Contains serine in position. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least 95% identical to at least 300 contiguous amino acids of SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least 95% identical to at least 300 contiguous amino acids of SEQ ID NO: 8, and the isolated polypeptide also has an amino acid sequence corresponding to position 352 of SEQ ID NO: 8. Contains serine in position. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least 98% identical to at least 300 contiguous amino acids of SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least 98% identical to at least 300 contiguous amino acids of SEQ ID NO: 8, and the isolated polypeptide also has an amino acid sequence corresponding to position 352 of SEQ ID NO: 8. Contains serine in position. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least 99% identical to at least 300 contiguous amino acids of SEQ ID NO:8. In some embodiments, the isolated polypeptide comprises or consists of an amino acid sequence that is at least 99% identical to at least 300 contiguous amino acids of SEQ ID NO: 8, and the isolated polypeptide also has an amino acid sequence corresponding to position 352 of SEQ ID NO: 8. Contains serine in position.

In some embodiments, the isolated polypeptide is at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, It contains or consists of at least about 70, at least about 80, at least about 90 or at least about 100 contiguous amino acids. In some embodiments, the isolated polypeptide also includes a serine at a position corresponding to position 352 of SEQ ID NO:8. In some embodiments, the isolated polypeptide is at least about 8, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 or at least about 100 contiguous amino acids and at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical Contains or consists of an amino acid sequence. In some embodiments, the isolated polypeptide also includes a serine at a position corresponding to position 352 of SEQ ID NO:8. In some embodiments, the isolated polypeptide is at least about 8, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 contiguous amino acids and at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences. In some embodiments, the isolated polypeptide also includes a serine at a position corresponding to position 352 of SEQ ID NO:8.

A representative wild-type B4GALT1 polypeptide sequence is shown in SEQ ID NO:7. A representative B4GALT1 variant polypeptide sequence is set forth in SEQ ID NO:8.

Isolated polypeptides disclosed herein may comprise the amino acid sequence of a naturally occurring B4GALT1 polypeptide, or may comprise a non-naturally occurring sequence. In some embodiments, naturally occurring sequences may differ from non-naturally occurring sequences due to conservative amino acid substitutions. For example, the sequences may be identical except for conservative amino acid substitutions.

In some embodiments, an isolated polypeptide disclosed herein is linked or fused to a heterologous polypeptide or heterologous molecule or label, various examples of which are disclosed elsewhere herein. For example, such proteins can be fused to heterologous polypeptides to provide increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N terminus, at the C terminus, or internally within the polypeptide. Fusion partners can help provide, for example, a T helper epitope (immunological fusion partner), or help express the protein in higher yields than the native recombinant polypeptide (expression enhancer). Specific fusion partners are both immunological fusion partners and expression enhancing fusion partners. Other fusion partners may be selected to increase the solubility of the polypeptide or to facilitate targeting of the polypeptide to the desired intracellular compartment. Some fusion partners contain an affinity tag, which facilitates purification of the polypeptide.

In some embodiments, the fusion protein is fused directly to a heterologous molecule or is linked to the heterologous molecule through a linker, such as a peptide linker. A suitable peptide linker sequence can be selected based on, for example, the following factors: 1) ability to adopt a flexible extended conformation; 2) resistance to adopting secondary structures that could interact with functional epitopes on the first and second polypeptides; and 3) lack of hydrophobic or charged residues that can react with the polypeptide functional epitope. For example, the peptide linker sequence may contain Gly, Asn, and Ser residues. Other nearly neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers are described, for example, in Maratea et al., Gene , 1985, 40, 39-46; Murphy et al., Proc. Natl. Acad. Sci. USA , 1986, 83, 8258-8262]; and U.S. Patent Nos. 4,935,233 and 4,751,180. The linker sequence may generally be, for example, from 1 to about 50 amino acids in length. If the first and second polypeptides have a non-essential N-terminal amino acid region that can be used to separate the functional domains and prevent steric hindrance, a linker sequence is generally not necessary.

In some embodiments, the polypeptide is operably linked to a cell-penetrating domain. For example, the cell-penetrating domain may be the HIV-1 TAT protein, the TLM cell-penetrating motif from human hepatitis B virus, MPG, Pep-1, VP22, a cell-penetrating peptide from herpes simplex virus, or a polyarginine peptide. It may be derived from a sequence (see, for example, International Patent No. WO2014/089290). The cell-penetrating domain may be located at the N-terminus, at the C-terminus, or anywhere within the protein.

In some embodiments, the polypeptide is operably linked to a heterologous polypeptide, such as a fluorescent protein, purification tag, or epitope tag, for ease of tracking or purification. Examples of fluorescent proteins include green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent protein (e.g., YFP , eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet , AmCyanl, Midoriishi-Cyan), red fluorescent protein (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred ), orange fluorescent protein (mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato), and any other suitable fluorescent protein. Examples of tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5. , AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV -G, histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin. In some embodiments, the heterologous molecule is an immunoglobulin Fc domain, peptide tag, transduction domain, poly(ethylene glycol), polysialic acid, or glycolic acid.

In some embodiments, the isolated polypeptide comprises a non-natural or modified amino acid or peptide analog. For example, there are a number of D-amino acids or amino acids with functional substitutions that differ from naturally occurring amino acids. Stereoisomers of peptide analogs as well as opposite stereoisomers of naturally occurring peptides are disclosed. These amino acids can be easily incorporated into a polypeptide chain by charging a tRNA molecule with the selected amino acid and inserting the analog amino acid into the peptide chain in a site-specific manner, for example, by engineering a genetic construct utilizing an amber codon.

In some embodiments, the isolated polypeptide is a peptide mimetic, which can be produced to resemble a peptide but is not linked through native peptide linkage. For example, linkages for amino acids or amino acid analogues are -CH ₂ NH-, -CH ₂ S-, -CH ₂ -, -CH=CH- (cis and trans), -COCH ₂ -, -CH(OH) Including, but not limited to, CH ₂ - and -CHH ₂ SO-. Peptide analogs may have more than one atom between bonding atoms, such as b-alanine, g-aminobutyric acid, etc. Amino acid analogs and peptide analogs usually have improved or desired properties, such as more economical production, higher chemical stability, improved pharmacological properties (half-life, absorption, potency, potency, etc.), altered specificity (e.g., broad biological activity), ), reduced antigenicity, and other desirable properties.

In some embodiments, the isolated polypeptide includes D-amino acids, which can be used to generate more stable peptides because D amino acids are not recognized by peptidases. Systematic substitution of one or more amino acids of the consensus sequence with a D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be advantageous in confining the peptide to a specific conformation (see, e.g., Rizo and Gierasch, Ann. Rev. Biochem ., 1992, 61, 387).

The disclosure also provides nucleic acid molecules encoding any of the polypeptides disclosed herein. This includes all degenerate sequences associated with a particular polypeptide sequence (i.e., all nucleic acids having a sequence encoding one particular polypeptide sequence as well as all nucleic acids, including degenerate nucleic acids encoding the disclosed variants and derivatives of a protein sequence) . Accordingly, although each specific nucleic acid sequence may not be recorded herein, each and every sequence is actually disclosed and described herein through the disclosed polypeptide sequence.

The disclosure also provides compositions comprising any one or more of the nucleic acid molecules and any one or more of the polypeptides disclosed herein. In some embodiments, the composition includes a carrier. In some embodiments, the carrier increases the stability of the nucleic acid molecule and/or polypeptide (e.g., given storage conditions where degradation products remain below a threshold, e.g., less than 0.5% by weight of the starting nucleic acid or protein (e.g., (e.g., -20°C, 4°C, or ambient temperature) to extend the period of time; or to increase in vivo stability). Examples of carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic acid-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. Including but not limited to these.

The disclosure also provides methods for producing any of the B4GALT1 polypeptides or fragments thereof provided herein. These B4GALT1 polypeptides or fragments thereof can be produced by any suitable method. For example, a B4GALT1 polypeptide or fragment thereof can be produced from a host cell comprising a nucleic acid molecule (e.g., a recombinant expression vector) encoding such B4GALT1 polypeptide or fragment thereof. This method involves culturing a host cell comprising a nucleic acid molecule (e.g., a recombinant expression vector) encoding a B4GALT1 polypeptide or fragment thereof under conditions sufficient to produce the B4GALT1 polypeptide or fragment thereof, thereby producing the B4GALT1 polypeptide or It may include producing fragments thereof. The nucleic acid can be operably linked to a promoter that is active in the host cell, and culturing can be performed under conditions under which the nucleic acid is expressed. Such methods may further include recovering the expressed B4GALT1 polypeptide or fragment thereof. Recovery may further include purifying the B4GALT1 polypeptide or fragment thereof.

Examples of suitable systems for protein expression include host cells, such as: bacterial cell expression systems (e.g. Escherichia coli , Lactococcus lactis), yeast cell expression systems (e.g. Saccharomyces cerevisiae, Pichia pastoris), insect cell expression systems (e.g. , baculovirus-mediated protein expression), and mammalian cell expression systems.

Examples of nucleic acid molecules encoding B4GALT1 polypeptides or fragments thereof are disclosed in more detail elsewhere herein. In some embodiments, the nucleic acid molecule is codon optimized for expression in a host cell. In some embodiments, the nucleic acid molecule is operably linked to a promoter that is active in the host cell. The promoter may be a heterologous promoter (i.e., a promoter other than the naturally occurring B4GALT1 promoter). Examples of suitable promoters for Escherichia coli include arabinose, lac , tac, and T7. Including, but not limited to, promoters. Examples of suitable promoters for Lactococcus lactis include, but are not limited to, the P170 and nisin promoters. Examples of suitable promoters for Saccharomyces cerevisiae include constitutive promoters such as alcohol dehydrogenase (ADHI) or enolase (ENO) promoters or inducible promoters such as PHO, CUP1, GAL1 and G10. Including but not limited to these. Examples of suitable promoters for Pichia pastoris include, but are not limited to, the alcohol oxidase I (AOX I) promoter, the glyceraldehyde 3 phosphate dehydrogenase (GAP) promoter, and the glutathione-dependent formaldehyde dehydrogenase (FLDI) promoter. Not limited. An example of a suitable promoter for baculovirus-mediated systems is the late viral strong polyhedrin promoter.

In some embodiments, the nucleic acid molecule encodes a tag in frame with the B4GALT1 polypeptide or fragment thereof to facilitate protein purification. Examples of tags are disclosed elsewhere herein. Such tags can bind to a partner ligand (e.g., immobilized on a resin) such that, for example, the tagged protein can be isolated from all other proteins (e.g., host cell proteins). Affinity chromatography, high performance liquid chromatography (HPLC), and size exclusion chromatography (SEC) are examples of methods that can be used to improve the purity of expressed proteins.

Other methods can also be used to produce B4GALT1 polypeptide or fragments thereof. For example, two or more peptides or polypeptides can be linked together by protein chemistry techniques. For example, peptides or polypeptides can be synthesized chemically using Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry. These peptides or polypeptides can be synthesized by standard chemical reactions. For example, a peptide or polypeptide may be synthesized from its synthetic resin and not cleaved, while other fragments of the peptide or protein may be synthesized and subsequently cleaved from the resin, thereby exposing functionally blocked end groups on these other fragments. It can be. By a peptide condensation reaction, these two fragments can be covalently linked via peptide bonds at their carboxyl and amino termini, respectively. Alternatively, the peptide or polypeptide can be synthesized independently in vivo as described herein. Once isolated, these independent peptides or polypeptides can be linked to form peptides or fragments thereof through similar peptide condensation reactions.

In some embodiments, enzymatic ligation of cloned or synthetic peptide segments allows relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or entire protein domains (Abrahmsen et al., Biochemistry , 1991 , 30, 4151). Alternatively, native chemical ligation of synthetic peptides can be utilized to construct large peptides or polypeptides by synthesis from shorter peptide fragments. This method may consist of a two-step chemical reaction (see Dawson et al., Science , 1994, 266, 776-779). The first step may be a chemoselective reaction of the unprotected synthetic peptide-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to provide a thioester-linked intermediate as the initial covalent product. Without changing the reaction conditions, these intermediates can undergo spontaneous and rapid intramolecular reactions forming native peptide bonds at the ligation site.

In some embodiments, unprotected peptide segments can be chemically linked, where the bond formed between these peptide segments as a result of chemical ligation is a non-natural (non-peptide) bond (Schnolzer et al., Science , 1992, 256 , 221]).

The disclosure also provides cells (e.g., recombinant host cells) comprising any one or more of the nucleic acid molecules and any one or more of the polypeptides disclosed herein. Cells may exist in vitro, ex vivo, or in vivo. Nucleic acid molecules can be linked to promoters and other regulatory sequences so that they can be expressed to produce the encoded protein.

In some embodiments, the cells are totipotent cells or pluripotent cells (e.g., embryonic stem (ES) cells, such as rodent ES cells, mouse ES cells, or rat ES cells). Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that retain the ability to develop into more than one differentiated cell type. Such pluripotent and/or totipotent cells may be, for example, ES cells or ES-like cells, such as induced pluripotent stem (iPS) cells. ES cells include embryo-derived totipotent or pluripotent cells that, upon introduction into the embryo, can contribute to any tissue of the developing embryo. ES cells can be derived from the inner cell mass of the blastocyst and can differentiate into any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

In some embodiments, the cell is a primary somatic cell, or a non-primary somatic cell. Somatic cells may include gametes, germ cells, germ cells, or any cell that is not an undifferentiated stem cell. In some embodiments, the cells can also be primary cells. Primary cells include cells or cultures of cells isolated directly from an organism, organ, or tissue. Primary cells include cells that have not been transformed or immortalized. Primary cells include any cells obtained from an organism, organ, or tissue that has not previously been passaged in tissue culture or has been previously passaged in tissue culture but cannot be passaged indefinitely in tissue culture. These cells can be isolated by conventional techniques and include, for example, somatic cells, hematopoietic cells, endothelial cells, epithelial cells, fibroblasts, mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclear cells, adipocytes, fat. Includes progenitor cells, neurons, glial cells, hepatocytes, skeletal myoblasts, and smooth muscle cells. For example, primary cells can be derived from connective tissue, muscle tissue, nervous system tissue, or epithelial tissue.

In some embodiments, cells are normally unable to proliferate indefinitely, but due to mutations or alterations, they can avoid normal cellular aging and instead continue to undergo division. These mutations or alterations may occur naturally or may be intentionally induced. Examples of immortalized cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g., HEK 293 cells), and mouse embryonic fibroblast cells (e.g., 3T3 cells). Many types of immortalized cells are well known. Immortalized or primary cells include cells typically used to culture or express recombinant genes or proteins. In some embodiments, the cell is a differentiated cell, such as a liver cell (eg, a human liver cell).

Cells may be derived from any source. For example, the cell may be a eukaryotic cell, an animal cell, a plant cell, or a fungal (e.g., yeast) cell. These cells may be fish cells or avian cells, or these cells may be mammalian cells, such as human cells, non-human mammalian cells, rodent cells, mouse cells or rat cells. Mammals include humans, non-human primates, monkeys, apes, cats, dogs, horses, bulls, deer, bison, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs), and livestock (e.g., bovine species). , such as cattle, steers, etc.; sheep species, such as sheep, goats, etc.; and porcine species, such as pigs and wild boars). Birds include, but are not limited to, chickens, turkeys, ostriches, geese, ducks, etc. Livestock and agricultural animals are also included. The term “non-human animal” excludes humans.

The present disclosure also provides methods for detecting the presence of a B4GALT1 variant gene, mRNA, cDNA, and/or polypeptide in a biological sample from a human subject. It is understood that within a population gene sequences, and the mRNA and proteins encoded by such genes, may vary due to polymorphisms, such as single-nucleotide polymorphisms. The sequences provided herein for the B4GALT1 gene, mRNA, cDNA and polypeptide are exemplary sequences only. Other sequences for the B4GALT1 gene, mRNA, cDNA and polypeptide are also possible.

A biological sample can be derived from any cell, tissue, or biological fluid from a subject. The sample may include any clinically relevant tissue, such as a bone marrow sample, tumor biopsy, fine needle aspirate, or body fluid such as blood, plasma, serum, lymph, ascites, cystic fluid, or urine. In some cases, the sample includes a buccal swab. Samples used in the methods disclosed herein will vary based on the assay format, nature of the detection method, and tissue, cell, or extract used as the sample. Biological samples can be processed differently depending on the assay used. For example, when detecting variant B4GALT1 nucleic acid molecules, preprocessing designed to isolate or enrich the sample for genomic DNA can be used. A variety of known techniques can be used for this purpose. When detecting levels of variant B4GALT1 mRNA, different techniques can be used to enrich biological samples with mRNA. A variety of methods can be used to detect the presence or level of mRNA or the presence of a specific variant genomic DNA locus.

In some embodiments, the present disclosure provides sequencing of at least a portion of a nucleic acid in a biological sample to determine whether the nucleic acid comprises nucleotides 53575 to 53577 of SEQ ID NO: 2 at a position corresponding to positions 53575 to 53577 of SEQ ID NO: 2. A method of detecting the presence or absence of a variant B4GALT1 nucleic acid molecule is provided, comprising the steps of:

In some embodiments, the present disclosure provides sequencing of at least a portion of a nucleic acid in a biological sample to determine whether the nucleic acid comprises nucleotides 1243-1245 of SEQ ID NO:4 at a position corresponding to positions 1243-1245 of SEQ ID NO:4. A method of detecting the presence or absence of a variant B4GALT1 nucleic acid molecule is provided, comprising the steps of:

In some embodiments, the present disclosure provides sequencing of at least a portion of a nucleic acid in a biological sample to determine whether the nucleic acid comprises nucleotides 1054-1056 of SEQ ID NO:6 at a position corresponding to positions 1054-1056 of SEQ ID NO:6. A method of detecting the presence or absence of a variant B4GALT1 nucleic acid molecule is provided, comprising the steps of:

In some embodiments, a method of detecting a variant B4GALT1 nucleic acid molecule (e.g., gene, mRNA, or cDNA) in a human subject comprises performing the assay on a biological sample from the human subject to determine if the nucleic acid molecule in the biological sample is SEQ ID NO: 8. It includes determining whether or not the nucleic acid sequence encoding serine is included at position 352. In some embodiments, the biological sample includes cells or cell lysates. This method includes, for example, obtaining a biological sample from a subject containing the B4GALT1 gene, mRNA or cDNA, and performing an assay on the biological sample to determine the gene at positions 53575 to 53577 (gene) of SEQ ID NO: 2, SEQ ID NO: 4, The position of the B4GALT1 gene, mRNA or cDNA corresponding to positions 1243 to 1245 (mRNA) or positions 1054 to 1056 (cDNA) of SEQ ID NO: 6 is serine instead of asparagine at the position corresponding to position 352 of the variant B4GALT1 polypeptide. It may include a step of determining whether to encrypt. Such assays may include, for example, determining the identity of this position on a particular B4GALT1 nucleic acid molecule.

In some embodiments, the assay comprises sequencing a portion of the B4GALT1 genomic sequence of a nucleic acid molecule in a biological sample from a human subject, wherein the portion sequenced comprises positions corresponding to positions 53575 to 53577 of SEQ ID NO:2. Including, analyzing the sequence; Sequencing a portion of the B4GALT1 mRNA sequence of a nucleic acid molecule in a biological sample from a human subject, wherein the portion sequenced comprises positions corresponding to positions 1243 to 1245 of SEQ ID NO: 4. ; or sequencing a portion of the B4GALT1 cDNA sequence of a nucleic acid molecule in a biological sample from a human subject, wherein the portion sequenced comprises positions corresponding to positions 1054 to 1056 of SEQ ID NO:6. Includes steps.

In some embodiments, the assay comprises: a) a biological sample comprising: i) a portion of the B4GALT1 genomic sequence adjacent to a position in the B4GALT1 genomic sequence corresponding to positions 53575-53577 of SEQ ID NO:2; ii) a portion of the B4GALT1 mRNA sequence adjacent to the position of B4GALT1 mRNA corresponding to positions 1243 to 1245 of SEQ ID NO: 4; or iii) contacting with a primer that hybridizes to a portion of the B4GALT1 cDNA sequence adjacent to the position of the B4GALT1 cDNA corresponding to positions 1054 to 1056 of SEQ ID NO:6; b) apply primers to at least i) a position in the B4GALT1 genomic sequence corresponding to positions 53575 to 53577; ii) the location of B4GALT1 mRNA corresponding to positions 1243 to 1245; or iii) extending through the position of B4GALT1 cDNA corresponding to positions 1054 to 1056; and c) the extension product of the primer encodes a serine at position 352 of SEQ ID NO: 8, i) a position corresponding to positions 53575 to 53577 of the B4GALT1 genome sequence; ii) a position corresponding to positions 1243 to 1245 of B4GALT1 mRNA; or iii) determining whether it contains a nucleotide at a position corresponding to positions 1054 to 1056 of the B4GALT1 cDNA. In some embodiments, only B4GALT1 genomic DNA is analyzed. In some embodiments, only B4GALT1 mRNA is analyzed. In some embodiments, only B4GALT1 cDNA is analyzed.

In some embodiments, the assay involves contacting a biological sample under stringent conditions with a primer or probe that specifically hybridizes to a variant B4GALT1 genomic sequence, mRNA sequence, or cDNA sequence rather than the corresponding wild-type B4GALT1 sequence and determines whether hybridization has occurred. It includes the step of determining whether or not.

In some embodiments, the assays described above include RNA sequencing (RNA-Seq). In some embodiments, the assay also includes reverse transcription polymerase chain reaction (RT-PCR).

In some embodiments, the methods utilize probes and primers of sufficient nucleotide length to bind to the target nucleic acid sequence and specifically detect and/or identify polynucleotides comprising the variant B4GALT1 gene, mRNA or cDNA. Hybridization conditions or reaction conditions can be determined by operating factors to achieve these results. This length may be any length sufficient for use with the detection method selected. Typically, for example, about 8, about 11, about 14, about 16, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 40, about 50, about 75, about 100, about 200, about 300, about 400, about 500, about 600 or about 700 nucleotides or more, or about 11 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, About 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700 or about 700 to about 800 or more Excess nucleotide lengths are used. These probes and primers can specifically hybridize to target sequences under highly stringent hybridization conditions. Although probes that are different from the target nucleic acid sequence but retain the ability to specifically detect and/or identify the target nucleic acid sequence can be designed by conventional methods, probes and primers must maintain complete nucleic acid sequence identity of the target sequence with adjacent nucleotides. You can have Accordingly, probes and primers are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, They may share about 98%, about 99% or 100% sequence identity or complementarity.

In some embodiments, specific primers are used to amplify the variant B4GALT1 locus and/or B4GALT1 variant mRNA or cDNA, which can be used as specific probes or themselves to identify the variant B4GALT1 locus in a biological sample or to amplify a specific B4GALT1 mRNA or cDNA. Amplicons can be produced that can be detected to determine the level of cDNA. The B4GALT1 variant locus can be used to refer to a genomic nucleic acid sequence comprising a position corresponding to positions 53575 to 53577 in SEQ ID NO:2. If the probe hybridizes to a nucleic acid molecule in a biological sample under conditions that permit binding of the probe to the nucleic acid molecule, this binding is detected and indicates the presence of a variant B4GALT1 locus or the presence or level of variant B4GALT1 mRNA or cDNA in the biological sample. can be allowed. This identification of bound probes is described. Particular probes are at least about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, and about 95% to about 100% identical (or may include complementary) sequences. Particular probes are at least about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, and about 95% to about 100% identical (or may include complementary) sequences. Particular probes are at least about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, and about 95% to about 100% identical (or may include complementary) sequences.

In some embodiments, to determine whether the nucleic acid complement of the biological sample comprises nucleotides encoding serine at positions 53575 to 53577 within the variant B4GALT1 gene locus (SEQ ID NO: 2), the biological sample is selected from the variant B4GALT1 gene locus (SEQ ID NO: 2). 2) To produce an amplicon diagnostic for the presence of a SNP at positions 53575 to 53577, a first primer derived from the 5' flanking sequence adjacent to positions 53575 to 53577 and the 3' flanking sequence adjacent to positions 53575 to 53577. It can be applied to a nucleic acid amplification method using a primer pair including a second primer derived from a flanking sequence. In some embodiments, amplicons may range in length from the sum of a primer pair plus one nucleotide base pair to any length of amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair, the limit of the amplification reaction, or about 20,000 nucleotide base pairs. Optionally, the primer pair comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides for positions 53575 to 53577 and each flanking positions 53575 to 53577. adjacent to the area. Similar amplicons can be generated from mRNA and/or cDNA sequences.

Representative methods for making and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd Ed., Vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989 (hereinafter “Sambrook et al., 1989”); Current Protocols in Molecular Biology , ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (updated regularly) (hereafter “Ausubel et al., 1992”); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990). PCR primer pairs can be analyzed, for example, by computer programs intended for this purpose, such as the PCR Primer Analysis Tool in Vector NTI Version 10 (Informax Inc., Bethesda, MD); PrimerSelect (DNASTAR Inc., Madison, WI, USA); and Primer3 (Version 0.4.0.COPYRGT., 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass., USA). Additionally, sequences can be scanned visually and primers can be identified manually using known guidelines.

As described in more detail below, any conventional nucleic acid hybridization or amplification or sequencing method can be used to specifically detect the presence of a variant B4GALT1 gene locus and/or levels of variant B4GALT1 mRNA or cDNA. In some embodiments, a nucleic acid molecule can be used as a primer to amplify a region of a B4GALT1 nucleic acid or hybridize the nucleic acid molecule to a nucleic acid molecule comprising a variant B4GALT1 gene locus or a nucleic acid molecule comprising variant B4GALT1 mRNA or cDNA under stringent conditions. It can be used as a probe.

A variety of nucleic acid techniques are known, including, for example, nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification. Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing.

Other methods include nucleic acid hybridization methods other than sequencing, including the use of labeled primers or probes (fluorescence in situ hybridization) directed against purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid can be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). . Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

Any method, including, for example, Hybridization Protection Assay (HPA), a quantitative assessment of a real-time amplification process, and determination of the amount of target sequence initially present in a sample but not based on real-time amplification, Can be used to detect non-amplified or amplified polynucleotides.

Nucleic acids are synthesized based on known methods of Southern (DNA:DNA) blot hybridization, in situ hybridization (ISH) and fluorescence in situ hybridization (FISH), for example, using appropriate probes, without necessarily requiring sequence amplification. Methods for identification are also provided. Southern blotting can be used to detect specific nucleic acid sequences. In this method, nucleic acids extracted from a sample are fragmented, electrophoretically separated in a matrix gel, and transferred to a membrane filter. Filter-bound nucleic acids are subjected to hybridization with labeled probes complementary to the sequence of interest. Hybridized probe bound to the filter is detected.

In hybridization techniques, stringent conditions can be used to ensure that a probe or primer will hybridize specifically to its target. In some embodiments , under stringent conditions, a polynucleotide primer or probe is directed to its target sequence (e.g., a variant B4GALT1 gene locus, mRNA or cDNA) will hybridize to a detectably greater degree at least 2-fold over background or 10-fold greater than background. Stringent conditions are sequence dependent and will be different in different situations. By controlling the stringency of hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homology probing). Alternatively, stringency conditions can be adjusted to allow for some mismatches in the sequence, such that a lower degree of identity is detected (heterogeneity probing). Typically, probes are less than about 1000 nucleotides long or less than about 500 nucleotides long.

Conditions of appropriate stringency that promote DNA hybridization, e.g., 6×sodium chloride/sodium citrate (SSC) at about 45°C, followed by washes of 2×SSC at 50°C, are known or described in Current Protocols in Molecular Biology. , John Wiley & Sons, NY (1989), 6.3.1-6.3.6]. Typically, stringent conditions for hybridization and detection are salt concentrations lower than about 1.5 M Na ion at pH 7.0 to 8.3, typically about 0.01 to 1.0 M Na ion concentration (or other salt), and short temperature probes (e.g. for longer probes (e.g., 10 to 50 nucleotides) and at least about 60°C for longer probes (e.g., 50 or more nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents, such as formamide. Exemplary low stringency conditions are hybridization with a buffer of 30-35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and 1× to 2× SSC (20× SSC) at 50-55°C. = 3.0M NaCl/0.3M trisodium citrate). Exemplary medium stringency conditions include hybridization in 40-45% formamide, 1.0M NaCl, 1% SDS at 37°C and washing in 0.5× to 1× SSC at 55-60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37°C, and washing in 0.1×SSC at 60-65°C. Optionally, the wash buffer may include about 0.1% to about 1% SDS. The period of hybridization is generally less than about 24 hours, typically about 4 to about 12 hours. The period of washing time will be at least a sufficient period of time to reach equilibrium.

In hybridization reactions, specificity is typically a function of post-hybridization washing, with the determining factors being the ionic strength and temperature of the final washing solution. For DNA-DNA hybrids, T _m is described in Meinkoth and Wahl, Anal. Biochem ., 1984, 138, 267-284 can be approximated from the equation: T _m = 81.5° C. + 16.6 (log M) + 0.41 (% GC) - 0.61 (% foam) - 500/L; where M is the molar concentration of the monovalent cation, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T _m is the temperature (under specified ionic strength and pH) at which 50% of the complementary target sequence hybridizes to the perfectly matched probe. T _m decreases by about 1°C for each 1% mismatch; Accordingly, T _m , hybridization and/or washing conditions can be adjusted to hybridize to the sequence of desired identity. For example, if sequences with greater than 90% identity are sought, T _m may be reduced by 10°C. Typically, stringent conditions are chosen to be about 5°C below the thermal melting point (T _m ) for a particular sequence and its complement at a defined ionic strength and pH. However, extremely stringent conditions may utilize hybridization and/or washing at temperatures 1°C, 2°C, 3°C, or 4°C lower than the thermal melting point (T _m ); Moderately stringent conditions may utilize hybridization and/or washing at temperatures 6°C, 7°C, 8°C, 9°C, or 10°C below the thermal melting point (T _m ); Low stringency conditions may utilize hybridization and/or washing at temperatures 11°C, 12°C, 13°C, 14°C, 15°C, or 20°C below the thermal melting point (T _m ). Using the above equations, hybridization and wash compositions, and desired T _m , one of ordinary skill in the art will understand that variations in the stringency of hybridization and/or wash solutions are implicitly described. If the desired degree of mismatch results in a T _m lower than 45°C (aqueous solution) or 32°C (formamide solution), it is optimal to increase the SSC concentration so that higher temperatures can be used.

Methods for detecting the presence and levels of variant B4GALT1 polypeptides in biological samples are also provided, including, for example, protein sequencing and immunoassays. In some embodiments, a method of detecting the presence of B4GALT1 Asn352Ser in a human subject comprises performing an assay on a biological sample from the human subject to determine the presence of B4GALT1 Asn352Ser in the biological sample.

Illustrative, non-limiting examples of protein sequencing techniques include, but are not limited to, mass spectrometry and Edman degradation. Exemplary immunoassays include, but are not limited to, immunoprecipitation, Western blot, immunohistochemistry, ELISA, immunocytochemistry, flow cytometry, and immuno-PCR. Polyclonal or monoclonal antibodies that are detectably labeled using a variety of known techniques (e.g., colorimetric, fluorescent, chemiluminescent, or radioactive) are suitable for use in immunoassays.

The present disclosure also provides methods for determining the susceptibility of a human subject to developing a cardiovascular condition or at risk of developing a cardiovascular condition. The subject can be any organism, including, for example, a human, non-human mammal, rodent, mouse or rat. In some embodiments, the method comprises detecting the presence of variant B4GALT1 genomic DNA, mRNA, or cDNA in a biological sample from a subject. It is understood that within a population, gene sequences and the mRNA encoded by such genes may vary due to polymorphisms, such as polymorphisms, such as SNPs. The sequences provided herein for the B4GALT1 gene, mRNA, cDNA and polypeptide are merely exemplary sequences, and other such sequences are also possible.

Non-limiting examples of cardiovascular conditions include increased levels of one or more serum lipids. Serum lipids include cholesterol, LDL, HDL, triglycerides, HDL-cholesterol, and non-HDL cholesterol or their subclasses (e.g., HDL2, HDL2a, HDL2b, HDL2c, HDL3, HDL3a, HDL3b, HDL3c, HDL3d, LDL1). , LDL2, LDL3, lipoprotein A, Lpa1, Lpa1, Lpa3, Lpa4 or Lpa5). Cardiovascular conditions may include increased levels of coronary artery calcification. Cardiovascular conditions may involve type IId glycosylation (CDG-IId). Cardiovascular conditions may include increased levels of fat around the heart. Cardiovascular conditions also include coronary artery disease (CAD), myocardial infarction (MI), peripheral artery disease (PAD), stroke, pulmonary embolism, deep vein thrombosis (DVT), and bleeding diathesis and coagulopathy. It can be included. Cardiovascular conditions may include atherothrombotic conditions. Atherothrombotic conditions may involve increased levels of fibrinogen. Atherothrombotic conditions may include fibrinogen-mediated blood clots. Cardiovascular conditions may involve increased levels of fibrinogen. Cardiovascular conditions may include fibrinogen-mediated blood clots. Cardiovascular conditions may include blood clots that form from involvement of fibrinogen activity. Fibrinogen-mediated blood clots, or blood clots formed from involvement of fibrinogen activity, can be present within any vein or artery within the body.

In some embodiments, a method of determining the susceptibility of a human subject to developing a cardiovascular condition comprises: a) performing an assay on a biological sample from the human subject to determine whether the nucleic acid molecule in the biological sample is of the full-length/mature variant B4GALT1 Asn352Ser polypeptide; determining whether the position corresponding to position 352 contains a nucleic acid sequence encoding serine; and b) if a nucleic acid molecule comprising a nucleic acid sequence encoding a serine at position 352 of the full-length/mature variant B4GALT1 Asn352Ser polypeptide is detected in the biological sample, the human subject is classified as having a reduced risk for the development of a cardiovascular condition. Alternatively, if a nucleic acid molecule comprising a nucleic acid sequence encoding a serine at position 352 of the full-length/mature variant B4GALT1 Asn352Ser polypeptide is not detected in the biological sample, the human subject is considered to be at increased risk for the development of a cardiovascular condition. Includes a classification step. In some embodiments, the variant B4GALT1 Asn352Ser polypeptide comprises SEQ ID NO:8. In some embodiments, the nucleic acid molecules in the biological sample are genomic DNA, mRNA, or cDNA.

In some embodiments, the present disclosure provides a method of determining the susceptibility of a human subject to developing a cardiovascular condition, the method comprising: a) performing an assay on a biological sample from the human subject to identify nucleic acid molecules in the biological sample; determining whether it includes nucleotides 53575 to 53577 of SEQ ID NO: 2 at a position corresponding to positions 53575 to 53577 of SEQ ID NO: 2; and b) if a nucleic acid molecule comprising nucleotides 53575 to 53577 of SEQ ID NO: 2 at a position corresponding to positions 53575 to 53577 of SEQ ID NO: 2 is detected in the biological sample, the human subject has a reduced risk for developing a cardiovascular condition. or, if a nucleic acid molecule comprising nucleotides 53575 to 53577 of SEQ ID NO: 2 at a position corresponding to positions 53575 to 53577 of SEQ ID NO: 2 is not detected in the biological sample, the human subject is at increased risk for the development of cardiovascular conditions. Includes the step of classifying it as having a risk.

In some embodiments, the present disclosure provides a method of determining the susceptibility of a human subject to developing a cardiovascular condition, the method comprising: a) performing an assay on a biological sample from the human subject to identify nucleic acid molecules in the biological sample; determining whether it includes nucleotides 1243 to 1245 of SEQ ID NO: 4 at a position corresponding to positions 1243 to 1245 of SEQ ID NO: 4; and b) if a nucleic acid molecule comprising nucleotides 1243 to 1245 of SEQ ID NO:4 at a position corresponding to positions 1243 to 1245 of SEQ ID NO:4 is detected in the biological sample, then the human subject is at reduced risk for developing a cardiovascular condition. classification, or if a nucleic acid molecule comprising nucleotides 1243 to 1245 of SEQ ID NO: 4 at a position corresponding to positions 1243 to 1245 of SEQ ID NO: 4 is not detected in the biological sample, the human subject is at increased risk for the development of a cardiovascular condition. It includes the step of classifying as having.

In some embodiments, the present disclosure provides a method of determining the susceptibility of a human subject to developing a cardiovascular condition, the method comprising: a) performing an assay on a biological sample from the human subject to identify nucleic acid molecules in the biological sample; determining whether it includes nucleotides 1054 to 1056 of SEQ ID NO:6 at a position corresponding to positions 1054 to 1056 of SEQ ID NO:6; and b) if a nucleic acid molecule comprising nucleotides 1054 to 1056 of SEQ ID NO:6 at a position corresponding to positions 1054 to 1056 of SEQ ID NO:6 is detected in the biological sample, then the human subject is at reduced risk for developing a cardiovascular condition. classification, or if a nucleic acid molecule comprising nucleotides 1054 to 1056 of SEQ ID NO:6 at positions corresponding to positions 1054 to 1056 of SEQ ID NO:6 is not detected in the biological sample, the human subject is at increased risk for the development of a cardiovascular condition. It includes the step of classifying as having.

In some embodiments, the method includes detecting the presence of variant B4GALT1 genomic DNA in a biological sample. In some embodiments, such methods include determining the susceptibility of a human subject to developing a cardiovascular condition or at risk of developing a cardiovascular condition, the method comprising: a) obtaining a biological sample from the subject comprising genomic DNA; b) performing an assay on genomic DNA to determine the identity of nucleotides within the DNA occupied positions corresponding to positions 53575 to 53577 of the variant B4GALT1 gene (e.g., see SEQ ID NO: 2); and c) classifying the subject as having a reduced risk for developing a cardiovascular condition if the position in the genomic DNA corresponding to positions 53575 to 53577 of the variant B4GALT1 gene encodes a serine rather than an asparagine. Alternatively, if the position in the genomic DNA corresponding to positions 53575 to 53577 of the variant B4GALT1 gene does not encode a serine rather than an asparagine, the subject may be classified as having an increased risk for the development of a cardiovascular condition.

In some embodiments, such methods include diagnosing a subject with a cardiovascular condition, comprising: a) obtaining a biological sample from the subject comprising genomic DNA; b) performing an assay on genomic DNA to determine the identity of nucleotides within the DNA occupied positions corresponding to positions 53575 to 53577 of the variant B4GALT1 gene (e.g., see SEQ ID NO: 2); and c) classifying the subject as having a cardiovascular condition if the position in the genomic DNA corresponding to positions 53575 to 53577 of the variant B4GALT1 gene encodes a serine rather than an asparagine. Alternatively, if the position in the genomic DNA corresponding to positions 53575 to 53577 of the variant B4GALT1 gene does not encode a serine but not an asparagine, the subject may be classified as not having a cardiovascular condition.

In some embodiments, the method includes detecting the presence of variant B4GALT1 mRNA in a biological sample. In some embodiments, such methods include determining the susceptibility of a human subject to developing a cardiovascular condition or at risk of developing a cardiovascular condition, the method comprising: a) obtaining a biological sample from the subject comprising mRNA; b) performing an assay on the mRNA to determine the identity of the nucleotides within the mRNA occupied positions corresponding to positions 1243 to 1245 of the variant B4GALT1 mRNA (see, e.g., SEQ ID NO: 4); and c) classifying the subject as having a reduced risk for developing a cardiovascular condition if the position in the mRNA corresponding to positions 1243 to 1245 of the variant B4GALT1 mRNA encodes a serine rather than an asparagine. Alternatively, if the position in the mRNA corresponding to positions 1243 to 1245 of the variant B4GALT1 mRNA does not encode a serine rather than an asparagine, the subject may be classified as having an increased risk for the development of a cardiovascular condition.

In some embodiments, such methods include diagnosing a subject with a cardiovascular condition, comprising: a) obtaining a biological sample from the subject comprising mRNA; b) performing an assay on the mRNA to determine the identity of the nucleotides within the mRNA occupied positions corresponding to positions 1243 to 1245 of the variant B4GALT1 mRNA (see, e.g., SEQ ID NO: 4); and c) classifying the subject as having a cardiovascular condition if the position in the mRNA corresponding to positions 1243 to 1245 of the variant B4GALT1 mRNA encodes a serine rather than an asparagine. Alternatively, if the position in the mRNA corresponding to positions 1243 to 1245 of the variant B4GALT1 mRNA does not encode a serine rather than an asparagine, the subject may be classified as not having a cardiovascular condition.

In some embodiments, the method includes detecting the presence of variant B4GALT1 cDNA in a biological sample. In some embodiments, such methods include determining the susceptibility of a human subject to developing a cardiovascular condition or at risk of developing a cardiovascular condition, the method comprising: a) obtaining a biological sample from the subject comprising cDNA; b) performing an assay on the cDNA to determine the identity of the nucleotides within the cDNA occupied positions corresponding to positions 1054 to 1056 of the variant B4GALT1 cDNA (see, e.g., SEQ ID NO:6); and c) if the position in the cDNA corresponding to positions 1054 to 1056 of the variant B4GALT1 cDNA encodes a serine rather than an asparagine, classifying the subject as having a reduced risk for developing a cardiovascular condition. Alternatively, if the position in the cDNA corresponding to positions 1054 to 1056 of the variant B4GALT1 cDNA does not encode a serine rather than an asparagine, the subject may be classified as having an increased risk for the development of a cardiovascular condition.

In some embodiments, such methods include diagnosing a subject with a cardiovascular condition, comprising: a) obtaining a biological sample from the subject comprising cDNA; b) performing an assay on the cDNA to determine the identity of the nucleotides within the cDNA occupied positions corresponding to positions 1054 to 1056 of the variant B4GALT1 cDNA (see, e.g., SEQ ID NO:6); and c) classifying the subject as having a cardiovascular condition if the position in the cDNA corresponding to positions 1054 to 1056 of the variant B4GALT1 cDNA encodes a serine and not an asparagine. Alternatively, if the position in the cDNA corresponding to positions 1054 to 1056 of the variant B4GALT1 cDNA does not encode a serine rather than an asparagine, the subject may be classified as not having a cardiovascular condition.

In some embodiments, the assay comprises sequencing a portion of the B4GALT1 genomic sequence of a nucleic acid molecule in a biological sample from a human subject, wherein the portion sequenced comprises positions corresponding to positions 53575 to 53577 of SEQ ID NO:2. Including, analyzing the sequence; Sequencing a portion of the B4GALT1 mRNA sequence of a nucleic acid molecule in a biological sample from a human subject, wherein the portion sequenced comprises positions corresponding to positions 1243 to 1245 of SEQ ID NO: 4. ; or sequencing a portion of the B4GALT1 cDNA sequence of a nucleic acid molecule in a biological sample from a human subject, wherein the portion sequenced comprises positions corresponding to positions 1054 to 1056 of SEQ ID NO:6. Includes steps.

In some embodiments, the assay involves a) a biological sample comprising: i) a portion of the B4GALT1 genomic sequence adjacent to a position in the B4GALT1 genomic sequence corresponding to positions 53575-53577 of SEQ ID NO:2; ii) a portion of the B4GALT1 mRNA sequence adjacent to the position of B4GALT1 mRNA corresponding to positions 1243 to 1245 of SEQ ID NO: 4; or iii) contacting with a primer that hybridizes to a portion of the B4GALT1 cDNA sequence adjacent to the position of the B4GALT1 cDNA corresponding to positions 1054 to 1056 of SEQ ID NO:6; b) apply primers to at least i) a position in the B4GALT1 genomic sequence corresponding to positions 53575 to 53577; ii) the location of B4GALT1 mRNA corresponding to positions 1243 to 1245; or iii) extending through the position of B4GALT1 cDNA corresponding to positions 1054 to 1056; and c) the extension product of the primer encodes a serine at position 352 of SEQ ID NO: 8, i) a position corresponding to positions 53575 to 53577 of the B4GALT1 genome sequence; ii) a position corresponding to positions 1243 to 1245 of B4GALT1 mRNA; or iii) determining whether it contains a nucleotide at a position corresponding to positions 1054 to 1056 of the B4GALT1 cDNA.

In some embodiments, the assay involves contacting a biological sample under stringent conditions with a primer or probe that specifically hybridizes to a variant B4GALT1 genomic sequence, mRNA sequence, or cDNA sequence rather than the corresponding wild-type B4GALT1 sequence and determines whether hybridization has occurred. It includes the step of determining whether or not. In some embodiments, the primer or probe specifically hybridizes to a position in the genomic DNA in the biological sample corresponding to positions 53575-53577 of SEQ ID NO:2. In some embodiments, the primer or probe specifically hybridizes to a position within the mRNA in the biological sample corresponding to positions 1243-1245 of SEQ ID NO:4. In some embodiments, the primer or probe specifically hybridizes to a position within the cDNA in the biological sample corresponding to positions 1054-1056 of SEQ ID NO:6.

Other assays that can be used in the methods disclosed herein include, for example, reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). Additional other assays that can be used in the methods disclosed herein include, for example, RNA sequencing (RNA-Seq) followed by determination of the presence or amount of variant mRNA or cDNA in a biological sample.

The disclosure also provides a method of determining the susceptibility of a human subject to developing a cardiovascular condition or diagnosing a subject with a cardiovascular condition, the method comprising: a) performing an assay on a biological sample from the human subject; determining whether the B4GALT1 polypeptide in the biological sample contains serine at a position corresponding to position 352 of SEQ ID NO: 8; and b) if a B4GALT1 polypeptide comprising a serine at a position corresponding to position 352 of SEQ ID NO. 8 is detected in the biological sample, the human subject is classified as having a reduced risk for developing a cardiovascular condition, or SEQ ID NO. If a B4GALT1 polypeptide comprising a serine at a position corresponding to position 352 of 8 is not detected in the biological sample, classifying the human subject as having an increased risk for developing a cardiovascular condition. In some embodiments, the method further includes obtaining a biological sample from the subject.

In some embodiments, when a subject is diagnosed as having a cardiovascular condition or as having an increased risk for developing a cardiovascular condition, a therapeutic or prophylactic agent that treats or prevents the cardiovascular condition is administered to the subject. Alternatively, the method may be used to treat one or more symptoms associated with progression to a more clinically advanced stage of cardiovascular disease, particularly in patients with increased LDL levels and/or with or at increased risk of thrombotic events. It may further include administering a therapeutic agent appropriate for prevention or alleviation.

The present disclosure provides nuclease agents, exogenous donor sequences, transcriptional activators, transcriptional repressors, antisense molecules such as antisense RNA, siRNA, and shRNA, B4GALT1 polypeptides or fragments thereof, and recombinant B4GALT1 genes or B4GALT1 polypeptides encoding A method of modifying a cell is provided by using any combination of expression vectors to express nucleic acids. The method may proceed in vitro, ex vivo, or in vivo. Nuclease agents, exogenous donor sequences, transcriptional activators, transcriptional repressors, antisense molecules such as antisense RNA, siRNA and shRNA, B4GALT1 polypeptide or fragments thereof, and expression vectors may be used in any form and as described elsewhere herein. They can be introduced into cells by any means, and all or some of them can be introduced simultaneously or sequentially in any combination. Some methods involve only altering the endogenous B4GALT1 gene within the cell. Some methods involve solely altering the expression of the endogenous B4GALT1 gene through the use of transcriptional activators or repressors or through the use of antisense molecules such as antisense RNA, siRNA and shRNA. Some methods involve solely introducing into cells a nucleic acid encoding a recombinant B4GALT1 gene or B4GALT1 polypeptide or fragment thereof. Some methods involve solely introducing a B4GALT1 polypeptide or fragment thereof into a cell (any one of the B4GALT1 polypeptides or fragments thereof disclosed herein or any combination thereof). Other methods include both altering the endogenous B4GALT1 gene within the cell and introducing into the cell a nucleic acid encoding a B4GALT1 polypeptide or fragment thereof or a recombinant B4GALT1 gene or B4GALT1 polypeptide or fragment thereof. Other methods include both altering the expression of the endogenous B4GALT1 gene within the cell and introducing into the cell a B4GALT1 polypeptide or fragment thereof or a nucleic acid encoding a recombinant B4GALT1 gene or B4GALT1 polypeptide or fragment thereof.

The present disclosure provides methods of modifying the endogenous B4GALT1 gene within a genome within a cell (e.g., a pluripotent cell or differentiated cell) through the use of a nuclease agent and/or an exogenous donor sequence. The method may proceed in vitro, ex vivo, or in vivo. Nuclease agents can be used alone or in combination with an exogenous donor sequence. Alternatively, exogenous donor sequences can be used alone or in combination with a nuclease agent.

Repair in response to double-strand breaks (DSBs) principally occurs through two conserved DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). (See Kasparek & Humphrey, Seminars in Cell & Dev. Biol. , 2011, 22, 886-897). Repair of a target nucleic acid (e.g., an endogenous B4GALT1 gene) mediated by an exogenous donor sequence may involve any exchange of genetic information between two polynucleotides. For example, NHEJ can also result in targeted integration of an exogenous donor sequence through direct ligation of the broken end with the end of the exogenous donor sequence (i.e., NHEJ-based capture). Repair can also occur through homology directed repair (HDR) or homologous recombination (HR). HDR or HR may require nucleotide sequence homology, use a "donor" molecule as a template for repair of a "target" molecule (i.e., a molecule that has suffered a double-strand break), and transfer genetic information from the donor to the target. Includes a form of nucleic acid repair that leads to the transfer of .

Targeted genetic modification to the endogenous B4GALT1 gene within the genome comprises a 5' homology arm that hybridizes to a 5' target sequence at a target genomic locus within the endogenous B4GALT1 gene and a 3' target sequence at the target genomic locus within the endogenous B4GALT1 gene. It can be generated by contacting an exogenous donor sequence that contains the 3' homology arm. The exogenous donor sequence can be recombined with the target genomic locus to create targeted genetic modifications to the endogenous B4GALT1 gene. As an example, the 5' homology arm may hybridize to the target sequence 5' at a position corresponding to positions 53575 to 53577 in SEQ ID NO: 1, and the 3' homology arm corresponds to positions 53575 to 53577 in SEQ ID NO: 1. It can hybridize to the target sequence 3' at the position. This method can, for example, result in a B4GALT1 gene containing a nucleotide sequence encoding serine at a position corresponding to position 352 of the full-length/mature polypeptide produced therefrom. Examples of exogenous donor sequences are disclosed elsewhere herein.

For example, a targeted genetic modification to an endogenous B4GALT1 gene in the genome may comprise a cell or the genome of a cell with one or more guide RNAs and a Cas protein that hybridizes to one or more guide RNA recognition sequences within a target genomic locus within the endogenous B4GALT1 gene. It can be created by contact. For example, such a method may include contacting the cell with a Cas protein and a guide RNA that hybridizes to a guide RNA recognition sequence within the endogenous B4GALT1 gene. In some embodiments, the guide RNA recognition sequence is located within a region corresponding to exon 5 of SEQ ID NO:1. In some embodiments, the guide RNA recognition sequence may comprise or is adjacent to positions corresponding to positions 53575 to 53577 of SEQ ID NO:1. For example, the guide RNA recognition sequence includes positions corresponding to positions 53575 to 53577 of SEQ ID NO: 1, or about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45 thereof. , about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides. As yet another example, the guide RNA recognition sequence may comprise or be adjacent to the start codon of the endogenous B4GALT1 gene or the stop codon of the endogenous B4GALT1 gene. For example, the guide RNA recognition sequence is within about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides of the start codon or stop codon. It can exist. The Cas protein and guide RNA form a complex, and the Cas protein cleaves the guide RNA recognition sequence. Cleavage by a Cas protein can produce a double-strand break or a single-strand break (e.g., if the Cas protein is a nickase). This method can result in an endogenous B4GALT1 gene with, for example, a region corresponding to exon 5 of SEQ ID NO:1 destroyed, a start codon destroyed, a stop codon destroyed, or the coding sequence deleted. Examples and variations of Cas (e.g., Cas9) proteins and guide RNAs that can be used in the methods are described elsewhere herein.

In some embodiments, two or more nuclease agents may be used. For example, two nuclease agents can be used, each targeting a nuclease recognition sequence within the region corresponding to exon 5 of SEQ ID NO: 1, or positions corresponding to positions 53575 to 53577 of SEQ ID NO: 1. (For example, about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about positions corresponding to positions 53575 to 53577 of SEQ ID NO: 1 30, about 25, about 20, about 15, about 10, or about 5 nucleotides). As another example, two or more nuclease agents may be used, each targeting a nuclease recognition sequence containing or adjacent to the start codon. As another example, two or more nuclease agents may be used, one targeting the nuclease recognition sequence containing or adjacent to the start codon, and one targeting the nuclease recognition sequence containing or adjacent to the stop codon. targeting, where cleavage by a nuclease agent may result in deletion of the coding region between the two nuclease recognition sequences. As yet another example, three or more nuclease agents may be used, one or more (e.g., two) targeting a nuclease recognition sequence comprising or adjacent to the start codon, and one or more (e.g., For example, type 2) targets a nuclease recognition sequence that includes or is adjacent to a stop codon, wherein cleavage by a nuclease agent includes a nuclease recognition sequence that includes or is adjacent to a start codon and a stop codon; This may result in deletion of the coding region between the adjacent nuclease recognition sequences.

In some embodiments, the cells may be further contacted with one or more additional guide RNAs that hybridize to additional guide RNA recognition sequences within the target genomic locus within endogenous B4GALT1 . By contacting the cell with one or more additional guide RNAs (e.g., a second guide RNA that hybridizes to a second guide RNA recognition sequence), cleavage by the Cas protein results in two or more double-strand breaks or two or more single-strand breaks (e.g., a second guide RNA that hybridizes to a second guide RNA recognition sequence). For example, if the Cas protein is a nickase), it can be produced.

In some embodiments, the cells may be further contacted with one or more exogenous donor sequences that recombine with the target genomic locus within the endogenous B4GALT1 gene to generate targeted genetic modifications. Examples and variations of exogenous donor sequences that can be used in the methods are disclosed elsewhere herein.

The Cas protein, guide RNA(s) and exogenous donor sequence(s) can be introduced into the cell in any form and by any means as described elsewhere herein, and the Cas protein, guide RNA(s) and All or part of the exogenous donor sequence(s) may be introduced simultaneously or sequentially in any combination.

In some embodiments, repair of a target nucleic acid (e.g., an endogenous B4GALT1 gene) by an exogenous donor sequence occurs through homologous direct repair (HDR). Homologous direct repair occurs when the Cas protein cleaves both strands of DNA within the endogenous B4GALT1 gene to create a double-strand break, while the Cas protein cleaves one strand of DNA within the target nucleic acid to create a single-strand break. This can occur, or when a Cas nickase is used to create a double strand break formed by two offset nicks. In this method, the exogenous donor sequence includes 5' and 3' homology arms corresponding to the 5' and 3' target sequences. The guide RNA recognition sequence(s) or cleavage site(s) are adjacent to the 5' target sequence, adjacent to the 3' target sequence, adjacent to both the 5' target sequence and the 3' target sequence, or adjacent to the 5' target sequence. and may not be adjacent to the 3' target sequence. In some embodiments, the exogenous donor sequence may further comprise a nucleic acid insert flanked by 5' and 3' homology arms, and the nucleic acid insert is inserted between the 5' and 3' target sequences. If a nucleic acid insert is not present, the exogenous donor sequence can serve to delete the genomic sequence between the 5' and 3' target sequences. Examples of exogenous donor sequences are disclosed elsewhere herein.

Alternatively, repair of the endogenous B4GALT1 gene mediated by an exogenous donor sequence can occur through non-homologous end joining (NHEJ)-mediated ligation. In this method, at least one end of the exogenous donor sequence comprises a short single-stranded region complementary to at least one overhang created by Cas-mediated cleavage in the endogenous B4GALT1 gene. Complementary ends in the exogenous donor sequence may flank the nucleic acid insert. For example, each end of the exogenous donor sequence may comprise a short single-stranded region complementary to an overhang created by Cas-mediated cleavage in the endogenous B4GALT1 gene, wherein this complementary region within the exogenous donor sequence is a nucleic acid It can be flanked on the insert.

Overhangs (i.e., staggered ends) can be created by excision of the blunt ends of double-strand breaks created by Cas-mediated cleavage. Although this excision may create a region of microhomology necessary for fragment joining, it may create unwanted or uncontrollable changes in the B4GALT1 gene. Alternatively, these overhangs can be created by using paired Cas nickases. For example, a cell can be contacted with a first nickase and a second nickase that cleave opposing strands of DNA, thereby modifying the genome through double nicking. This involves activating the cell with a first Cas protein nickase, a first guide RNA that hybridizes to a first guide RNA recognition sequence within the target genomic locus within the endogenous B4GALT1 gene, a second Cas protein nickase, and a second guide RNA within the target genomic locus within the endogenous B4GALT1 gene. This can be accomplished by contacting a second guide RNA that hybridizes to the guide RNA recognition sequence. The first Cas protein and the first guide RNA form a first complex, and the second Cas protein and the second guide RNA form a second complex. The first Cas protein nickase cleaves the first strand of genomic DNA within the first guide RNA recognition sequence, and the second Cas protein nickase cleaves the second strand of genomic DNA within the second guide RNA recognition sequence, and optionally The exogenous donor sequence recombines with the target genomic locus within the endogenous B4GALT1 gene to create a targeted genetic modification.

The first nickase cleaves the first strand of genomic DNA (i.e., the complementary strand), and the second nickase cleaves the second strand of genomic DNA (i.e., the non-complementary strand). The first nickase and the second nickase are mutated, for example, by mutating catalytic residues in the RuvC domain of Cas9 (e.g., the D10A mutation described elsewhere herein) or by mutating catalytic residues in the HNH domain of Cas9. mutating (e.g., the H840A mutation described elsewhere herein). In this method, double nicking can be used to create double strand breaks with staggered ends (i.e., overhangs). The first guide RNA and the second guide RNA recognition sequence are positioned on the first and second strands of the DNA to create a cleavage site such that the nick created by the first nickase and the second nickase creates a double strand break. It can be. Overhangs are created when nicks within the first and second CRISPR RNA recognition sequences are offset. The offset window may be, for example, at least about 5 bp, at least about 10 bp, at least about 20 bp, at least about 30 bp, at least about 40 bp, at least about 50 bp, at least about 60 bp, at least about 70 bp, at least about 80 bp, at least about 90 bp, at least about It may be 100 bp or more (see, e.g., Ran et al., Cell , 2013, 154, 1380-1389; Mali et al., Nat. Biotech. , 213, 31, 833-838; and Shen et al. al., Nat. Methods , 2014, 11, 399-404].

Various types of targeted genetic modifications can be introduced using the methods described herein. Such targeted modifications may include, for example, the addition of one or more nucleotides, the deletion of one or more nucleotides, the substitution of one or more nucleotides, point mutations, or combinations thereof. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 7, at least 8, at least 9 or at least 10 or more nucleotides are altered (e.g., deleted, inserted or substituted). Targeted genomic modifications can be made.

These targeted genetic modifications can result in disruption of the target genomic locus. Disruptions may include alterations in regulatory elements (e.g., promoters or enhancers), missense mutations, nonsense mutations, frame-shift mutations, truncation mutations, null mutations, or insertions or deletions of a small number of nucleotides (e.g. , resulting in frameshift mutations), which may result in inactivation (i.e., loss of function) or loss of allele. For example, a targeted modification may involve destruction of the start codon of the endogenous B4GALT1 gene such that the start codon is no longer functional.

In some embodiments, the targeted modification may comprise a deletion between the first and second guide RNA recognition sequences or between the Cas cleavage site. If an exogenous donor sequence (e.g., a repair template or targeting vector) is used, modifications may include deletions between the first and second guide RNA recognition sequences or between the Cas cleavage site, as well as deletions between the 5' target sequence and the 3' target sequence. It may involve the insertion of nucleic acid inserts between target sequences.

In some embodiments, when an exogenous donor sequence is used alone or in combination with a nuclease agent, the modifications include deletions between the 5' target sequence and the 3' target sequence, as well as deletions between the first and second homologous chromosomes. may involve the insertion of a nucleic acid insert between the 5' and 3' target sequences in the pair, thereby resulting in a homozygously modified genome. Alternatively, if the exogenous donor sequence includes a 5' homology arm and a 3' homology arm without a nucleic acid insert, the modification may involve a deletion between the 5' target sequence and the 3' target sequence.

A deletion between a first guide RNA recognition sequence and a second guide RNA recognition sequence or a deletion between a 5' target sequence and a 3' target sequence means that the deleted nucleic acid consists only of the nucleic acid sequence between the first and second nuclease cleavage sites. It may be an accurate deletion if it consists only of the nucleic acid sequence between the 5' target sequence and the 3' target sequence and there is no additional deletion or insertion at the modified genomic target locus. Deletions between the first guide RNA recognition sequence and the second guide RNA recognition sequence may also be imprecise deletions extending past the first and second nuclease cleavage sites, which may be imprecise by non-homologous end joining (NHEJ). Consistent with a repair, it may result in additional deletions and/or insertions at the altered genomic locus. For example, the deletion may be about 1 bp, about 2 bp, about 3 bp, about 4 bp, about 5 bp, about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 100 bp, It may extend about 200 bp, about 300 bp, about 400 bp, about 500 bp, or more. Likewise, altered genomic loci may have additional insertions consistent with incorrect repair by NHEJ, e.g., about 1 bp, about 2 bp, about 3 bp, about 4 bp, about 5 bp, about 10 bp, about 20 bp, about 30 bp, about 40 bp, It may include an insertion of about 50 bp, about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, or more.

The targeted genetic modification may be, for example, a biallelic modification or a monoallelic modification. Biallelic modifications include events where the same modification occurs at the same locus on a corresponding homologous chromosome (e.g., in a diploid cell), or where different modifications occur at the same locus on a corresponding homologous chromosome. In some embodiments, the targeted genetic modification is a monoallelic modification. Monoallelic variants include events in which the variant occurs for only one allele (i.e., for the endogenous B4GALT1 gene on only one of the two homologous chromosomes). Homologous chromosomes include chromosomes that have identical genes at the same locus but possibly different alleles (for example, chromosomes that pair up during meiosis).

Monoallelic mutations can result in cells that are heterozygous for the targeted endogenous B4GALT1 variant. Heterozygosity includes the situation where only one allele of the B4GALT1 gene (i.e., the corresponding allele on two homologous chromosomes) has a targeted modification.

Biallelic variants can result in homozygosity for the targeted variant. Homozygosity includes the situation where both alleles of the B4GALT1 gene (i.e., corresponding alleles on two homologous chromosomes) have the targeted variant. Alternatively, a biallelic modification may result in compound heterozygosity (e.g., hemizygosity) for the targeted modification. Compound heterozygosity occurs when both alleles at a target locus (i.e., alleles on two homologous chromosomes) are modified, but in different ways (e.g., a targeted modification in one allele and inactivation of the other allele, or destruction) situation.

The methods disclosed herein may further include the step of identifying cells with a modified endogenous B4GALT1 gene. A variety of methods can be used to identify cells with a targeted genetic modification, such as a deletion or insertion. Such methods may include identifying one cell with a targeted genetic modification within the B4GALT1 gene. Screening can be performed to identify these cells with altered genomic loci. The screening step may include quantitative assays (e.g., loss of allele (LOA) and/or gain of allele (GOA) assays) to assess the allelic modification (MOA) of the parental chromosome.

Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparison of immobilized probe(s), INVADER ^™ probes, TAQMAN ^™ Molecular Beacon probes, or ECLIPSE ^™ probe technologies. Includes genomic hybridization, constant temperature DNA amplification, and quantitative hybridization. Conventional assays for screening for targeted modifications, such as long-range PCR, Southern blotting or Sanger sequencing, can also be used. These assays are typically used to obtain evidence for linkage between the inserted targeting vector and the targeted genomic locus. For example, for a long-range PCR assay, one primer may recognize a sequence within the inserted DNA, while the other primer recognizes a target genomic locus sequence past the end of the homology arm of the targeting vector.

Next generation sequencing (NGS) can also be used for screening. Next-generation sequencing may also be referred to as “NGS” or “massively parallel sequencing” or “high throughput sequencing.” In some embodiments, screening for targeted cells using a selection marker is not necessary. For example, the MOA and NGS assays described herein may rely on not using a selection cassette.

The present disclosure also provides methods of altering the expression of a nucleic acid encoding a B4GALT1 polypeptide. In some embodiments, expression is altered through cleavage with a nuclease agent to cause destruction of the nucleic acid encoding the endogenous B4GALT1 polypeptide, as described in more detail elsewhere herein. In some embodiments, expression is altered through the use of a DNA-binding protein fused to or linked to a transcriptional activation domain or a transcriptional repression domain. In some embodiments, expression is altered through the use of RNA interference compositions, such as antisense RNA, shRNA, or siRNA.

In some embodiments, expression of a nucleic acid encoding an endogenous B4GALT1 gene or B4GALT1 polypeptide may cause the cell or the genome within the cell to be nicked or duplicated at a recognition sequence at a target genomic locus within the endogenous B4GALT1 gene or nucleic acid encoding the endogenous B4GALT1 polypeptide. It can be modified by contacting it with a nuclease agent that induces strand breaks. Such truncation may result in disruption of the expression of the endogenous B4GALT1 gene or nucleic acid encoding the endogenous B4GALT1 polypeptide. For example, the nuclease recognition sequence can include or be adjacent to the start codon of the endogenous B4GALT1 gene. For example, the recognition sequence may be within about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides of the start codon, and Cleavage by clease agents can destroy the start codon. In some embodiments, two or more nuclease agents may be used, each targeting a nuclease recognition sequence that includes or is adjacent to the start codon. In some embodiments, two or more nuclease agents may be used, one targeting a nuclease recognition sequence containing or adjacent to the start codon, and one targeting a nuclease recognition sequence containing or adjacent to the stop codon. targeting, where cleavage by a nuclease agent may result in deletion of the coding region between the two nuclease recognition sequences. In some embodiments, three or more nuclease agents may be used, one or more (e.g., two) targeting a nuclease recognition sequence comprising or adjacent to the start codon, and one or more (e.g., , 2 types) target a nuclease recognition sequence containing or adjacent to a stop codon, where cleavage by a nuclease agent targets a nuclease recognition sequence containing or adjacent to the start codon and a nuclease recognition sequence containing or adjacent to the stop codon. This may result in deletion of the coding region between the nuclease recognition sequences. Other examples of modifying the endogenous B4GALT1 gene or nucleic acid encoding the endogenous B4GALT1 polypeptide are disclosed elsewhere herein.

In some embodiments, the expression of an endogenous B4GALT1 gene or a nucleic acid encoding an endogenous B4GALT1 polypeptide can be modified by contacting the cell or the genome within the cell with a DNA-binding protein that binds to a target genomic locus within the endogenous B4GALT1 gene. The DNA-binding protein may be, for example, a nuclease-inactive Cas protein fused to a transcriptional activator domain or a transcriptional repressor domain. Other examples of DNA-binding proteins include zinc finger proteins fused to a transcriptional activator domain or a transcriptional repressor domain, or transcriptional activator-like effector (TALE) proteins fused to a transcriptional activator domain or a transcriptional repressor domain. . Examples of such proteins are disclosed elsewhere herein.

Recognition sequences for DNA-binding proteins (e.g., guide RNA recognition sequences) may be present anywhere within the endogenous B4GALT1 gene or nucleic acid encoding the B4GALT1 polypeptide suitable for modifying expression. In some embodiments, the recognition sequence may be within a regulatory element, such as an enhancer or promoter, or may be adjacent to a regulatory element. For example, the recognition sequence may include or be adjacent to the start codon of the endogenous B4GALT1 gene. In some embodiments, the recognition sequence may be within about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides of the start codon.

In some embodiments, antisense molecules can be used to alter the expression of the endogenous B4GALT1 gene or nucleic acid encoding a B4GALT1 polypeptide. Examples of antisense molecules include, but are not limited to, antisense RNA, siRNA, and shRNA. These antisense RNAs, siRNAs or shRNAs can be designed to target any region of the mRNA. For example, antisense RNA, siRNA, or shRNA can be designed to target unique regions of B4GALT1 mRNA.

Nucleic acids and proteins disclosed herein can be introduced into cells by any means. In some embodiments, introduction may be accomplished by any means, and one or more components (e.g., two of the components, or all components) may be introduced into the cell simultaneously or sequentially in any combination. . For example, the exogenous donor sequence may be introduced before introduction of the nuclease agent, or may be introduced after introduction of the nuclease agent (e.g., the exogenous donor sequence may be introduced before or after introduction of the nuclease agent). may be administered at approximately 1, 2, 3, 4, 8, 12, 24, 36, 48, or 72 hours). Contacting the genome of a cell with a nuclease agent or an exogenous donor sequence involves the addition of one or more nuclease agents or a nucleic acid encoding a nuclease agent (e.g., one or more Cas proteins or one or more Cas proteins). A nucleic acid that encodes one or more guide RNAs or one or more guide RNAs (i.e., one or more CRISPR RNAs and one or more tracrRNAs) and/or one or more exogenous donor sequences into the cell. can do. Contacting the genome of a cell (i.e., contacting the cell) may include introducing only one, more than one, or all of the above components into the cell.

The nuclease agent can be introduced into the cell in the form of a protein or in the form of a nucleic acid encoding the nuclease agent, such as RNA (e.g., messenger RNA (mRNA)) or DNA. When introduced in the form of DNA, the DNA can be operably linked to a promoter that is active in the cell. Such DNA may be present in one or more expression constructs.

In some embodiments, the Cas protein is a protein, e.g., in the form of a Cas protein complexed with a gRNA, or in the form of a nucleic acid, e.g., RNA (e.g., messenger RNA (mRNA)) or DNA encoding the Cas protein. It can be introduced into cells in this form. Guide RNA can be introduced into cells in the form of RNA or in the form of DNA encoding the guide RNA. When introduced in the form of DNA, the DNA encoding the Cas protein and/or guide RNA can be operably linked to a promoter that is active in the cell. Such DNA may be present in one or more expression constructs. For example, such expression constructs may be components of a single nucleic acid molecule. Alternatively, it may be isolated from any combination of two or more types of nucleic acid molecules (i.e., DNA encoding one or more CRISPR RNAs, DNA encoding more than one type of tracrRNA, and DNA encoding Cas proteins as separate may be a component of a nucleic acid molecule).

In some embodiments, DNA encoding nuclease agents (e.g., Cas proteins and guide RNAs) and/or DNA encoding exogenous donor sequences can be introduced into cells via DNA minicircles. DNA minicircles are supercoiled DNA molecules that can be used for non-viral gene transfer without an origin of replication or an antibiotic selection marker. Therefore, DNA minicircles are typically smaller in size than plasmid vectors. This DNA is devoid of bacterial DNA and therefore lacks the unmethylated CpG motifs found in bacterial DNA.

The methods described herein do not depend on the specific method of introducing the nucleic acid or protein into the cell, but simply provide the nucleic acid or protein access to the interior of at least one cell. Methods for introducing nucleic acids and proteins into various cell types are known and include, but are not limited to, stable transfection methods, transient transfection methods, and virus-mediated methods.

Transfection protocols as well as protocols for introducing nucleic acids or proteins into cells can vary. Non-limiting transfection methods include liposomes; nanoparticles; Calcium, dendrimer; and chemical-based transfection methods using cationic polymers such as DEAE-dextran or polyethyleneimine. Non-chemical methods include electroporation, sono-poration, and optical transfection. Particle-based transfection involves the use of gene guns, or magnet-assisted transfection. Viral methods can also be used for transfection.

Introduction of nucleic acids or proteins into cells can also be done by electroporation, by intracytoplasmic injection, by viral infection, by adenovirus, by adeno-associated virus, by lentivirus, by retrovirus, by transfection. It may be mediated by injection, by lipid-mediated transfection, or by nucleofection. Nucleofection is an improved electroporation technique that allows nucleic acid substrates to be delivered into the nucleus through the nuclear membrane as well as into the cytoplasm. Additionally, the use of nucleofection in the methods disclosed herein typically results in a much smaller number of cells than regular electroporation (e.g., only about 2 million compared to 7 million by regular electroporation). in need. In some embodiments, nucleofection is performed using the ^{LONZA® NUCLEOFECTOR® system} .

Introduction of nucleic acids or proteins into cells can also be achieved by microinjection. Microinjection of mRNA is usually injected into the cytoplasm (e.g., to deliver the mRNA directly to the translation machinery), whereas microinjection of DNA encoding a protein or Cas protein is usually injected into the nucleus. Alternatively, microinjection may be performed by injection into both the nucleus and the cytoplasm: the needle may first be introduced into the nucleus and the first amount may be injected, and the second amount may be injected into the cytoplasm as the needle is removed from the cell. It can be injected into the body. When a nuclease agonist protein is injected into the cytoplasm, the protein may contain a nuclear localization signal to ensure delivery to the nucleus/pronucleus.

Methods for introducing nucleic acids or proteins into cells include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediated delivery. or implantable device-mediated delivery. Methods for modifying cells in vivo by administering nucleic acids or proteins to a subject are disclosed elsewhere herein. Introduction of nucleic acids and proteins into cells can also be achieved by hydrodynamic delivery (HDD).

Methods for introducing nucleic acids or proteins into cells include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediated delivery. or implantable device-mediated delivery. In some embodiments, the nucleic acid or protein is carried in a carrier, e.g., poly(lactic acid) (PLA) microspheres, poly(D,L-lactic acid-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, Lipid cochleates, or lipids may be introduced into cells within microtubules.

Introduction of a nucleic acid or protein into a cell may be performed once or multiple times over a period of time. In some embodiments, the introduction occurs at least 2 times over a period of time, at least 3 times over a period of time, at least 4 times over a period of time, at least 5 times over a period of time, at least 6 times over a period of time, or at least over a period of time. at least 7 times over a period of time, at least 8 times over a period of time, at least 9 times over a period of time, at least 10 times over a period of time, at least 11 times over a period of time, at least 12 times over a period of time, at least 13 times over a period of time, at least 14 times over a period of time, at least 15 times over a period of time, at least 16 times over a period of time, at least 17 times over a period of time, at least 18 times over a period of time, at least 19 times over a period of time or at least a period of time. It can be performed at least 20 times.

In some embodiments, the cells used in the methods and compositions have DNA stably incorporated into the genome. In such cases, contacting may involve providing cells with the construct already stably incorporated into the genome. In some embodiments, cells used in the methods disclosed herein may have a pre-existing Cas-encoding gene stably incorporated into the genome (i.e., Cas-ready cells). In some embodiments, the polynucleotide is integrated into the cell's genome and can be inherited by its progeny. Any protocol can be used for stable incorporation of the various components of a DNA construct or targeted genomic integration system.

Any nuclease agent that induces a nick or double-strand break within the desired recognition sequence or any DNA-binding protein that binds the desired recognition sequence can be used in the methods and compositions disclosed herein. Naturally occurring or native nuclease agents may be used, as long as the nuclease agent induces a nick or double-strand break in the desired recognition sequence. Likewise, naturally occurring or native DNA-binding proteins can be used, as long as they bind to the desired recognition sequence. Alternatively, modified or engineered nuclease agents or DNA-binding proteins may be used. An engineered nuclease agent or DNA-binding protein can be derived from a native, naturally occurring nuclease agent or DNA-binding protein, or it can be artificially created or synthesized. An engineered nuclease agent or DNA-binding protein can recognize a recognition sequence, for example, wherein the recognition sequence is recognized by a native (unengineered or unmodified) nuclease agent or DNA-binding protein. It's not a priority. Modifications of a nuclease agent or DNA-binding protein can be as small as one amino acid in a protein cleaving agent or one nucleotide in a nucleic acid cleaving agent.

Recognition sequences for nuclease agents include DNA sequences in which a nick or double-strand break is induced by the nuclease agent. Likewise, the recognition sequence for a DNA-binding protein includes the DNA sequence to which the DNA-binding protein will bind. The recognition sequence may be endogenous (or native) to the cell or the recognition sequence may be exogenous to the cell. The recognition sequence may also be exogenous to the desired polynucleotide of interest placed at the target locus. In some embodiments, the recognition sequence occurs only once in the genome of the host cell.

Active variants and fragments of the exemplified recognition sequences are also provided. Such active variants are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to a given recognition sequence. %, at least 96%, at least 97%, at least 98% or at least 99% or 100% sequence identity, wherein the active variant retains biological activity and can be cleaved by a nuclease agent in a sequence-specific manner. Can be recognized and cut. Assays for measuring double-strand breaks in recognition sequences by nuclease agents are known (e.g., TAQMAN ^™ qPCR assay, Frendewey et al., Methods in Enzymology , 2010, 476, 295-307). .

The length of the recognition sequence may vary, for example, the recognition sequence, TALE, is about 30 to 36 bp for a zinc finger protein or zinc finger nuclease (ZFN) pair (i.e., about 15 to 18 bp for each ZFN). A recognition sequence that is approximately 36 bp for a protein or transcription activator-like effector nuclease (TALEN), or a recognition sequence that is approximately 20 bp for a CRISPR/Cas9 guide RNA.

The recognition sequence of the DNA-binding protein or nuclease agent may be located anywhere at or near the target genomic locus. The recognition sequence may be located within the coding region of a gene (eg, the B4GALT1 gene) or within a regulatory region that affects expression of the gene. The recognition sequence of a DNA-binding protein or nuclease agent may be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region.

One type of DNA-binding protein that can be used in the various methods and compositions disclosed herein is TALE. TALEs may be fused or linked to, for example, an epigene modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Examples of such domains are described below in relation to Cas proteins and can also be found, for example, in International Patent No. WO 2011/145121. Correspondingly, one type of nuclease agent that can be used in the various methods and compositions disclosed herein is TALEN. Transcription-activator-like (TAL) effector nucleases are a class of sequence-specific nucleases that can be used to create double strand breaks in specific target sequences in the genome of a prokaryotic or eukaryotic organism. TAL effector nucleases are produced by fusing a native or engineered TAL effector or functional portion thereof to the catalytic domain of an endonuclease such as FokI . The uniquely modular TAL effector DNA binding domain potentially allows the design of proteins with any given DNA recognition specificity. Accordingly, the DNA binding domain of a TAL effector nuclease can be engineered to recognize a specific DNA target site and thus used to create a double strand break in the desired target sequence. Examples of suitable TAL nucleases, and methods of making suitable TAL nucleases, see, for example, US Patent Publication No. 2011/0239315; No. 2011/0269234; No. 2011/0145940; No. 2003/0232410; No. 2005/0208489; No. 2005/0026157; No. 2005/0064474; No. 2006/0188987; and 2006/0063231.

In some TALENs, each monomer of the TALEN contains 33 to 35 TAL repeats that recognize a single base pair through two hypervariable residues. In some TALENs, the nuclease agonist is a chimeric protein containing a TAL-repeat-based DNA binding domain operably linked to an independent nuclease, such as the FokI endonuclease. For example, the nuclease agent may comprise a first TAL-repeat-based DNA binding domain and a second TAL-repeat-based DNA binding domain, wherein the first and second TAL-repeat-based Each DNA binding domain is operably linked to a FokI nuclease, wherein the first and second TAL-repeat-based DNA binding domains bind target DNA separated by spacer sequences of varying length (about 12 to about 20 bp). Recognizing two adjacent target DNA sequences on each strand of the sequence, the FokI nuclease subunit dimerizes to produce an active nuclease that creates a double-strand break in the target sequence.

Another example of a DNA-binding protein is the zinc finger protein. These zinc finger proteins can be fused or linked to, for example, an epigene modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Examples of such domains are described below in relation to Cas proteins and can also be found, for example, in International Patent No. WO 2011/145121. Correspondingly, another example of a nuclease agent that can be used in the various methods and compositions disclosed herein is ZFN. In some ZFNs, each monomer of the ZFN contains three or more zinc finger-based DNA binding domains, where each zinc finger-based DNA binding domain binds a 3bp subsite. In other ZFNs, the ZFN is a chimeric protein containing a zinc finger-based DNA binding domain operably linked to an independent nuclease, such as the FokI endonuclease. For example, the nuclease agent may comprise a first ZFN and a second ZFN, wherein each of the first ZFN and the second ZFN is operably linked to a FokI nuclease subunit, and wherein the first ZFN and the second ZFN are each operably linked to a FokI nuclease subunit. 2 ZFNs recognize two adjacent target DNA sequences on each strand of the target DNA sequence separated by a spacer of approximately 5 to 7 bp, where the FokI nuclease subunit dimerizes to produce an active nuclease that creates a double-strand break. Create.

Other suitable DNA-binding proteins and nuclease agents for use in the methods and compositions described herein include the CRISPR-Cas system described elsewhere herein.

DNA-binding proteins or nuclease agents can be introduced into cells by any known means. Polypeptides encoding DNA-binding proteins or nuclease agents can be introduced directly into cells. Alternatively, polynucleotides encoding DNA-binding proteins or nuclease agents can be introduced into cells. When a polynucleotide encoding a DNA-binding protein or nuclease agent is introduced into a cell, the DNA-binding protein or nuclease agent may be expressed transiently, conditionally, or constitutively within the cell. For example, a polynucleotide encoding a DNA-binding protein or nuclease agent can be contained in an expression cassette and operably linked to a conditional promoter, inducible promoter, constitutive promoter, or tissue-specific promoter. . These promoters are discussed in more detail elsewhere herein. In some embodiments, the DNA-binding protein or nuclease agonist may be introduced into the cell as an mRNA encoding the DNA-binding protein or nuclease agonist.

A polynucleotide encoding a DNA-binding protein or nuclease agent can be stably integrated into the genome of a cell and can be operably linked to a promoter active in the cell. Alternatively, the polynucleotide encoding the DNA-binding protein or nuclease agent may be present within the targeting vector or in a vector or plasmid separate from the targeting vector containing the insert polynucleotide.

When a DNA-binding protein or nuclease agent is provided to a cell through introduction of a polynucleotide encoding the DNA-binding protein or nuclease agent, the polynucleotide encoding such DNA-binding protein or nuclease agent Compared to naturally occurring polynucleotides encoding DNA-binding proteins or nuclease agents, they can be modified to substitute codons that are used at a higher frequency in the cells of interest. In some embodiments, a polynucleotide encoding a DNA-binding protein or nuclease agent is selected from a given prokaryotic or eukaryotic cell of interest, such as a bacterial cell, yeast cell, human cell, non-human cell, mammalian cell, rodent cell, It can be modified to substitute codons that are used at a higher frequency when compared to naturally occurring polynucleotide sequences in mouse cells, rat cells or any other host cell of interest.

The methods disclosed herein can utilize the Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)/CRISPR-Associated (Cas) system or components of such systems to modify the genome within a cell. there is. The CRISPR-Cas system includes transcripts and other elements involved in directing the expression, or activity, of Cas genes. CRISPR-Cas systems may be Type I, Type II, or Type III systems. Alternatively, the CRISPR/Cas system may be, for example, a type V system (e.g., subtype V-A or subtype V-B). The methods and compositions disclosed herein can utilize the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-directed cleavage of nucleic acids.

The CRISPR-Cas system used in the methods disclosed herein does not occur in nature. For example, some CRISPR/Cas systems use non-naturally occurring CRISPR complexes containing gRNA and Cas proteins that do not naturally occur together.

Cas proteins generally contain at least one RNA recognition or binding domain that can interact with a guide RNA (gRNA, described in more detail below). Cas proteins may also include a nuclease domain (e.g., a DNase or RNase domain), a DNA binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and other domains. The nuclease domain possesses catalytic activity for cleavage of nucleic acids, including the breaking of covalent bonds in nucleic acid molecules. Cleavage may produce blunt or staggered ends and may be single or double stranded. Wild-type Cas9 protein will typically produce blunt cleavage products. Alternatively, wild-type Cpf1 protein (e.g., FnCpf1) may result in a cleavage product with a 5-nucleotide 5' overhang, with cleavage occurring after the 18th base pair from the PAM sequence on the non-targeting strand and after 23 base pairs on the targeted strand. Occurs after the second base. The Cas protein may have global cleavage activity to create a double strand break in the endogenous B4GALT1 gene (e.g., a double strand break with blunt ends), or it may be a nickase that creates a single strand break in the endogenous B4GALT1 gene. there is.

Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Casl0d, CasF , CasG, CasH, Csy1, Csy2, Csy3, Cse1(CasA), Cse2(CasB), Cse3(CasE), Cse4(CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cu1966, and homologs or modified versions thereof. Including but not limited to these.

In some embodiments, the Cas protein is a Cas9 protein or is derived from a Cas9 protein from a type II CRISPR-Cas system. Cas9 proteins are derived from type II CRISPR-Cas systems, or typically share four major motifs with a conserved structure. Motifs 1, 2 and 4 are RuvC-like motifs and motif 3 is a HNH motif. Exemplary Cas9 proteins include Streptococcus pyogenes, Streptococcus thermophilus , Streptococcus sp. , Staphylococcus aureus , and Nocardiopsis dassonvillei , Streptomyces pristinaespiralis, Streptomyces viridochromogenes , Streptomyces viridochromogenes , Streptomyces Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides , Bacillus selenitireducens selenitireducens ), Exiguobacterium sibiricum , Lactobacillus delbrueckii, Lactobacillus salivarius , Microscilla marina , Burkholderiales bacterium bacterium ), Polaromonas naphthalenivorans, Polaromonas sp. , Crocosphaera watsonii, Cyanothece sp. , Microcystis Aeruginosa ( Microcystis aeruginosa ), Synechococcus sp. ), Acetohalobium arabaticum , Ammonifex degensii , Caldicelulosiruptor becscii, Candidatus Desulforudis , Clostridium botulinum ( Clostridium botulinum ), Clostridium difficile ( Clostridium difficile ), Finegoldia magna (Natranaerobius thermophilus), Pelotomaculum thermopropionicum ( Pelotomaculum thermopropionicum ), Asidi Acidithiobacillus caldus , Acidithiobacillus ferrooxidans, Allochromatium vinosum , Marinobacter sp. , Nitrosococcus halophilus , Nitrosococcus watsoni , Pseudoalteromonas haloplanktis , Ktedonobacter racemifer, Methanohalobium evestigatum, Anavar Anabaena variabilis , Nodularia spumigena, Nostoc sp. , Arthrospira maxima , Arthrospira platensis , Arthrospira sp . , Lyngbya sp. , Microcoleus chthonoplastes, Oscillatoria sp. ), Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina . Additional examples of Cas9 family members are described in International Patent No. WO 2014/131833. Cas9 from S. pyogenes (assigned SwissProt accession number Q99ZW2) is an exemplary enzyme. Cas9 from S. aureus (assigned UniProt accession number J7RUA5) is another exemplary enzyme.

Another example of a Cas protein is the Cpf1 (CRISPR from Prevotella and Francisella 1) protein. Cpf1 is a large protein (approximately 1300 amino acids) containing a RuvC-like nuclease domain homologous to the corresponding domain of Cas9 along with a counterpart to the characteristic arginine-rich cluster of Cas9. However, Cpf1 lacks the HNH nuclease domain present in the Cas9 protein, and the RuvC-like domain is continuous in the Cpf1 sequence, in contrast to Cas9, which contains a long insert containing the HNH domain. Exemplary Cpf1 proteins include Francisella tularensis 1, Francisella tularensis subsp. novicida , Prevotella albensis , Lachnospiraceae Bacterium ( Lachnospiraceae bacterium ) MC2017 1, Butyrivibrio proteoclasticus , Peregrinibacteria bacterium GW2011_GWA2_33_10 , Parcubacteria bacterium GW2011_GWC2_44_17 , Mitella sp . . ) SCADC, Acidaminococcus sp. BV3L6 , Lachnospiraceae bacterium MA2020 , Candidatus Methanoplasma termitum , Eubacterium eligens ), Moraxella bovoculi 237, Leptospira inadai , Lachnospiraceae bacterium ND2006 , Porphyromonas crevioricanis 3 , Prevotella Including, but not limited to, those from Prevotella disiens , and Porphyromonas macacae. Cpf1 (FnCpf1; assigned UniProt accession number A0Q7Q2) from Francisella novicida U112 is an exemplary enzyme.

The Cas protein may be a wild-type protein (i.e., that which occurs in nature), a modified Cas protein (i.e., a Cas protein variant), or a fragment of a wild-type or modified Cas protein. The Cas protein may also be an active variant or fragment of a wild-type or modified Cas protein. The active variant or fragment is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96% of the wild-type or modified Cas protein or portion thereof. %, at least 97%, at least 98% or at least 99% or 100% sequence identity, wherein the active variant retains the ability to cleave at the desired cleavage site and thus nick-induced or double-strand break-induced Remains active. Assays for nick-inducing or double-strand break-inducing activity are known and generally measure the overall activity and specificity of Cas proteins for DNA substrates containing cleavage sites.

Cas proteins may include at least one nuclease domain, such as a DNase domain. For example, the wild-type Cpf1 protein generally contains a RuvC-like domain that cleaves both strands of target DNA, possibly in dimeric form. Cas proteins may also include at least two nuclease domains, such as DNase domains. For example, wild-type Cas9 proteins generally contain a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains can each cleave a different strand of double-stranded DNA to create a double-strand break in the DNA.

Cas proteins (e.g., nuclease-active Cas proteins or nuclease-inactive Cas proteins) can also be operably linked to heterologous polypeptides as fusion proteins. For example, a Cas protein can be fused to a cleavage domain, epigene modification domain, transcriptional activation domain, or transcriptional repressor domain. Examples of transcriptional activation domains include the herpes simplex virus VP16 activation domain, VP64 (which is a tetrameric derivative of VP16), NFκB p65 activation domain, p53 activation domains 1 and 2, CREB (cAMP response element binding protein) activation domain, E2A activation domain, and Contains an NFAT (nuclear factor of activated T-cell) activation domain. Other examples include Oct1, Oct-2A, SP1, AP-2, CTF1, P300, CBP, PCAF, SRC1, PvALF, ERF-2, OsGAI, HALF-1, C1, AP1, ARF-5, ARF-6, Activation domains from ARF-7, ARF-8, CPRF1, CPRF4, MYC-RP/GP, TRAB1PC4 and HSF1 (US Patent Application Publication No. 2016/0237456, European Patent No. EP3045537 and See PCT Publication No. WO 2011/145121).

In some embodiments, a transcriptional activation system comprising a dCas9-VP64 fusion protein coupled to MS2-p65-HSF1 can be used. In these systems, the guide RNA can be designed with an aptamer sequence attached to the sgRNA tetraloop and stem-loop 2 designed to bind to the dimerized MS2 bacteriophage coat protein (see, e.g., Konermann et al., Nature , 2015, 517, 583-588]). Examples of transcriptional repressor domains include the inducible cAMP early repressor (ICER) domain, Kruppel-associated box A (KRAB-A) repressor domain, YY1 glycine-rich repressor domain, Sp1-like repressor, and E(spl) repressor. factor, ΙκB inhibitor, and MeCP2. Other examples include transcriptional repressor domains from A/B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, SID4X, MBD2, MBD3, DNMT1, DNMG3A, DNMT3B, Rb, and ROM2. However, it is not limited to these (e.g. European Patent No. EP3045537 and PCT Publication No. WO 2011/145121). Cas proteins can also be fused to heterologous polypeptides to provide increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N terminus, at the C terminus, or internally within the Cas protein.

Cas proteins are Cas proteins fused to heterologous polypeptides that provide intracellular localization. Such heterologous polypeptides may include, for example, one or more nuclear localization signals (NLS), such as SV40 NLS for targeting to the nucleus, a mitochondrial localization signal for targeting to mitochondria, an ER retention signal, etc. This intracellular localization signal can be located at the N-terminus, at the C-terminus, or anywhere within the protein. The NLS may contain a stretch of basic amino acids and may be a monopartite sequence or a bipartite sequence.

Cas proteins can also be operably linked to a cell-penetrating domain. For example, the cell-penetrating domain may be the HIV-1 TAT protein, the TLM cell-penetrating motif from human hepatitis B virus, MPG, Pep-1, VP22, a cell-penetrating peptide from herpes simplex virus, or a polyarginine peptide. It can be derived from a sequence. The cell-penetrating domain can be located at the N-terminus, at the C-terminus, or anywhere within the Cas protein.

Cas proteins can also be operably linked to heterologous polypeptides, such as fluorescent proteins, purification tags, or epitope tags for ease of tracking or purification. Examples of fluorescent proteins include green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent protein (e.g., YFP , eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet , AmCyanl, Midoriishi-Cyan), red fluorescent protein (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred ), orange fluorescent protein (mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato) and any other suitable fluorescent protein. Examples of tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5. , AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV -G, histidine (His), biotin carboxyl carrier protein (BCCP) and calmodulin.

Cas9 protein can also be tethered to an exogenous donor sequence or labeled nucleic acid. This tethering (i.e., physical connection) can be achieved through covalent or non-covalent interactions, and tethering can be direct (e.g., by modification of cysteine or lysine residues on the protein or by intein modification). This can be achieved through direct fusion or chemical conjugation), or through one or more intervening linkers or adapter molecules such as streptavidin or aptamers. Noncovalent strategies for synthesizing protein-nucleic acid conjugates include biotin-streptavidin and nickel-histidine methods. Covalent protein-nucleic acid conjugates can be synthesized by linking appropriately functionalized nucleic acids and proteins using a wide range of chemistries. Some of these chemistries involve direct attachment of oligonucleotides to amino acid residues (e.g., lysine amines or cysteine thiols) on the protein surface, while other, more complex methods involve post-translational modification of the protein or catalytic or reactive protein domains. inclusion is required. Methods for covalent attachment of proteins to nucleic acids may include, for example, chemical cross-linking of oligonucleotides to protein lysine or cysteine residues, ligation of expressed proteins, chemoenzymatic methods, and the use of photoaptamers. You can. The exogenous donor sequence or labeled nucleic acid can be tethered to the C-terminus, to the N-terminus, or to an internal region within the Cas9 protein. In some embodiments, the exogenous donor sequence or labeled nucleic acid is tethered to the C-terminus or N-terminus of the Cas9 protein. Likewise, the Cas9 protein can be tethered to the 5' end, 3' end, or internal region within an exogenous donor sequence or labeled nucleic acid. In some embodiments, the Cas9 protein is tethered to an exogenous donor sequence or to the 5' end or 3' end of a labeled nucleic acid.

Cas proteins can be provided in any form. For example, the Cas protein can be provided in the form of a protein, such as a Cas protein complexed with a gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid encoding the Cas protein, such as RNA (e.g., messenger RNA (mRNA)) or DNA. In some embodiments, nucleic acids encoding Cas proteins can be codon optimized for efficient translation into proteins in specific cells or organisms. For example, the nucleic acid encoding the Cas protein can be expressed as a naturally occurring polynucleotide in a bacterial cell, yeast cell, human cell, non-human cell, mammalian cell, rodent cell, mouse cell, rat cell, or any other host cell of interest. Compared to the nucleotide sequence, it can be modified to replace codons that are used with higher frequency. When a nucleic acid encoding a Cas protein is introduced into a cell, the Cas protein may be transiently, conditionally, or constitutively expressed in the cell.

A nucleic acid encoding a Cas protein can be stably integrated into the genome of a cell and operably linked to a promoter active in the cell. Alternatively, a nucleic acid encoding a Cas protein can be operably linked to a promoter in an expression construct. Expression constructs include any nucleic acid construct capable of directing the expression of a gene or other nucleic acid sequence of interest (e.g., a Cas gene) and delivering such nucleic acid sequence of interest to a target cell. For example, a nucleic acid encoding a Cas protein can be present in a targeting vector containing a nucleic acid insert and/or a vector containing DNA encoding a gRNA. Alternatively, it may be present in a vector or plasmid separate from the targeting vector containing the nucleic acid insert and/or separate from the vector containing the DNA encoding the gRNA. Promoters that can be used in expression constructs include, for example, eukaryotic cells, human cells, non-human cells, mammalian cells, non-human mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, rabbit cells. , pluripotent cells, embryonic stem (ES) cells, or zygotes. Such promoters may be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. In some embodiments, the promoter may be a bidirectional promoter that directs both expression of the Cas protein in one direction and expression of the guide RNA in the other direction. Such a bidirectional promoter can consist of: 1) a complete, conventional promoter containing three external control elements: a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box; , unidirectional Pol III promoter; and 2) a second basic Pol III promoter containing a PSE and a TATA box fused to the 5' end of the DSE in reverse orientation. For example, in the H1 promoter, the DSE is adjacent to the PSE and TATA boxes, and the promoter can be made bidirectional by creating a hybrid promoter where transcription in the reverse direction is controlled by appending the PSE and TATA boxes from the U6 promoter. The use of a bidirectional promoter to express the genes encoding the Cas protein and guide RNA simultaneously allows the creation of compact expression cassettes to facilitate delivery.

The present disclosure also provides a guide RNA (gRNA) that binds to a Cas protein (e.g., Cas9 protein) and targets the Cas protein to a specific location within a target DNA (e.g., the B4GALT1 gene). In some embodiments, the guide RNA is effective to direct the Cas enzyme to bind to or cleave the endogenous B4GALT1 gene, wherein the guide RNA includes, for example, positions 53575 to 53577 of SEQ ID NO: 1 or adjacent thereto. It comprises a DNA-targeting segment that hybridizes to a guide RNA recognition sequence within the endogenous B4GALT1 gene. For example, the guide RNA recognition sequence is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about positions 53575 to 53577 of SEQ ID NO: 1. It may be within 100, about 200, about 300, about 400, about 500 or about 1,000 nucleotides. Another exemplary guide RNA includes a DNA-targeting segment that hybridizes to a guide RNA recognition sequence in the endogenous B4GALT1 gene within the region corresponding to exon 5 of SEQ ID NO:1. Other exemplary guide RNAs include DNA-targeting segments that hybridize to a guide RNA recognition sequence in the endogenous B4GALT1 gene that includes or is adjacent to the start codon of the endogenous B4GALT1 gene or that includes or is adjacent to the stop codon of the endogenous B4GALT1 gene. For example, the guide RNA recognition sequence is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300 of the start codon. , within about 400, about 500, or about 1,000 nucleotides, or about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about a stop codon. It may be within 200, about 300, about 400, about 500 or about 1,000 nucleotides. The endogenous B4GALT1 gene can be a B4GALT1 gene from any organism. For example, the endogenous B4GALT1 gene can be the human B4GALT1 gene or an ortholog from another organism, such as a non-human mammal, rodent, mouse, or rat.

In some embodiments, the guide RNA recognition sequence is at the 5' end of the human B4GALT1 gene. In some embodiments, the guide RNA recognition sequence is adjacent to the transcription start site (TSS) of the human B4GALT1 gene. In some embodiments, the guide RNA recognition sequence is at the 3' end of the human B4GALT1 gene. In some embodiments, the guide RNA recognition sequence is adjacent to positions 53575-53577 of SEQ ID NO:1. Exemplary guide RNA recognition sequences adjacent to positions 53575 to 53577 of SEQ ID NO: 1 include ATTAGTTTTTAGAGGCATGT (SEQ ID NO: 9) and GGCTCTCAGGCCAAGTGTAT (SEQ ID NO: 10) (all 5' to positions 53575 to 53577 of SEQ ID NO: 1) and TACTCCTTCCCCCTTTAGGA ( SEQ ID NO: 11) and GTCCGAGGCTCTGGGCCTAG (SEQ ID NO: 12) (all 3' to positions 53575 to 53577 of SEQ ID NO: 1).

The guide RNA may include two segments: a DNA-targeting segment and a protein-binding segment. Some gRNAs may comprise two separate RNA molecules: an activator-RNA (e.g., tracrRNA) and a target-RNA (e.g., CRISPR RNA or crRNA). Other gRNAs are also single RNA molecules (single RNA polynucleotides; single-molecule gRNA, single-guide RNA or sgRNA). For Cas9, for example, the single-guide RNA may comprise a crRNA fused to a tracrRNA (e.g., via a linker). For Cpf1, for example, only crRNA is required to achieve cleavage. gRNAs include both bi-molecular (i.e. modular) gRNAs and single-molecule gRNAs.

The DNA-targeting segment (crRNA) of a given gRNA contains a nucleotide sequence complementary to a sequence in the target DNA (i.e., guide RNA recognition sequence). The DNA-targeting segment of the gRNA interacts with the target DNA (e.g., the B4GALT1 gene) in a sequence-specific manner through hybridization (i.e., base pairing). As such, the nucleotide sequence of the DNA-targeting segment can vary and determines the location within the target DNA at which the gRNA and target DNA will interact. The DNA-targeting segment of the gRNA of interest can be modified to hybridize to any desired sequence within the target DNA. Naturally occurring crRNAs vary depending on the CRISPR/Cas system and organism, but often have a targeting segment of about 21 to about 72 nucleotides in length, flanked by two direct repeats (DRs) of about 21 to about 46 nucleotides in length. Contains. For S. pyogenes, the DR is 36 nucleotides long and the targeting segment is 30 nucleotides long. The 3' positioned DR is complementary to the corresponding tracrRNA, hybridizes with it, and eventually binds to the Cas protein.

The DNA-targeting segment is at least about 12 nucleotides, at least about 15 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides. It may have a length of at least about 35 nucleotides, or at least about 40 nucleotides. This DNA-targeting segment can be about 12 nucleotides to about 100 nucleotides, about 12 nucleotides to about 80 nucleotides, about 12 nucleotides to about 50 nucleotides, about 12 nucleotides to about 40 nucleotides, about 12 nucleotides. It may have a length of from about 30 nucleotides, from about 12 nucleotides to about 25 nucleotides, or from about 12 nucleotides to about 20 nucleotides. For example, the DNA targeting segment may be about 15 nucleotides to about 25 nucleotides (e.g., about 17 nucleotides to about 20 nucleotides or about 17 nucleotides, about 18 nucleotides, about 19 nucleotides or about 20 nucleotides). nucleotides) (see, e.g., US Patent Publication No. 2016/0024523). For Cas9 from S. pyogenes, a typical DNA-targeting segment is about 16 to about 20 nucleotides long or about 17 to about 20 nucleotides long. For Cas9 from S. aureus, a typical DNA-targeting segment is about 21 to about 23 nucleotides long. For Cpf1, a typical DNA-targeting segment is at least about 16 nucleotides long or at least about 18 nucleotides long.

The percent complementarity between the guide RNA recognition sequence and the DNA-targeting sequence in the target DNA is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%. , at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. The percent complementarity between the guide RNA recognition sequence in the target DNA and the DNA-targeting sequence may be at least about 60% over about 20 contiguous nucleotides. As an example, the percent complementarity between the guide RNA recognition sequence in the target DNA and the DNA-targeting sequence is about 100% over about 14 contiguous nucleotides at the 5' end of the guide RNA recognition sequence within the complementary strand of the target DNA, and about 100% over the remainder. It is 0%. In this case, the DNA-targeting sequence can be considered to be approximately 14 nucleotides in length. As another example, the percent complementarity between the guide RNA recognition sequence in the target DNA and the DNA-targeting sequence is about 100% over the 7 contiguous nucleotides at the 5' end of the guide RNA recognition sequence in the complementary strand of the target DNA, and over the remainder. It's about 0%. In this case, the DNA-targeting sequence can be considered to be approximately 7 nucleotides in length. In some guide RNAs, at least about 17 nucleotides within the DNA-target sequence are complementary to the target DNA. For example, the DNA-targeting sequence may be about 20 nucleotides long and may contain 1, 2, or 3 matches with the target DNA (guide RNA recognition sequence). In some embodiments, the mismatch is not adjacent to a protospacer adjacent motif (PAM) sequence (e.g., the mismatch is at the 5' end of the DNA-targeting sequence, or the mismatch is adjacent to the PAM sequence). At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 base pairs away).

Guide RNAs may contain modifications or sequences that provide additional desirable characteristics (e.g., modified or controlled stability; intracellular targeting; tracking with a fluorescent label; binding sites for proteins or protein complexes, etc.). Examples of such modifications include, for example, 5' caps (e.g., 7-methylguanylate cap (m7G)); 3' polyadenylated tail (i.e., 3' poly(A) tail); riboswitch sequences (e.g., to allow controlled stability and/or controlled accessibility by proteins and/or protein complexes); stability control sequence; Sequences that form dsRNA duplexes (i.e., hairpins); Modifications or sequences that target RNA to an intracellular location (e.g., nucleus, mitochondria, chloroplast, etc.); Modifications or sequences that provide tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescence detection, sequences that allow fluorescence detection, etc.); Providing a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.) modification or sequence; and combinations thereof.

Guide RNA may be provided in any form. For example, the gRNA can be provided in the form of RNA, as two molecules (separate crRNA and tracrRNA) or as one molecule (sgRNA), and optionally in the form of a complex with a Cas protein. For example, gRNA can be prepared by in vitro transcription using, for example, T7 RNA polymerase. Guide RNA can also be prepared by chemical synthesis.

gRNA can also be provided in the form of DNA encoding the gRNA. DNA encoding a gRNA may encode a single RNA molecule (sgRNA) or separate RNA molecules (e.g., separate crRNA and tracrRNA). In the latter case, the DNA encoding the gRNA may be provided as one DNA molecule or as separate DNA molecules encoding the crRNA and tracrRNA, respectively. If the gRNA is provided in the form of DNA, the gRNA may be expressed transiently, conditionally, or constitutively in the cell. DNA encoding a gRNA can be stably integrated into the genome of a cell and operably linked to a promoter active in the cell. Alternatively, DNA encoding the gRNA can be operably linked to a promoter in an expression construct. For example, DNA encoding a gRNA may be present in a vector containing a heterologous nucleic acid. The vector may further comprise an exogenous donor sequence and/or the vector may further comprise a nucleic acid encoding a Cas protein. Alternatively, the DNA encoding the gRNA may be present in a vector or plasmid that is separate from the vector containing the exogenous donor sequence and/or the vector containing the nucleic acid encoding the Cas protein. Promoters that can be used in such expression constructs include, for example, eukaryotic cells, human cells, non-human cells, mammalian cells, non-human mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, rabbits. and a promoter that is active in one or more of the cells, pluripotent cells, embryonic stem cells, or zygotes. Such promoters may be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. Such promoters may also be, for example, bidirectional promoters. Specific examples of suitable promoters include RNA polymerase III promoter, such as human U6 promoter, rat U6 polymerase III promoter or mouse U6 polymerase III promoter.

The present disclosure also provides guidance for one or more guide RNAs disclosed herein (e.g., 1, 2, 3, 4 or more guide RNAs) and methods for increasing the stability of an isolated nucleic acid molecule or protein (e.g. , extending the period under given storage conditions (e.g., -20°C, 4°C, or ambient temperature) during which degradation products remain below a threshold, e.g., less than 0.5% by weight of the starting nucleic acid or protein; or in vivo. Provides a composition comprising a carrier (increasing stability). Examples of such carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic acid-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microspheres. Including, but not limited to, tubes. Such compositions may further include a Cas protein, such as a Cas9 protein or a nucleic acid encoding a Cas protein. Such compositions may include one or more (e.g., 1, 2, 3, 4 or more) exogenous donor sequences and/or one or more (e.g., 1, 2, 3, or more) exogenous donor sequences as disclosed elsewhere herein. It may further comprise 4 or more targeting vectors and/or one or more (e.g., 1, 2, 3, 4 or more) expression vectors.

The guide RNA recognition sequence includes a nucleic acid sequence present in the target DNA (e.g., B4GALT1 gene) to which the DNA-targeting segment of the gRNA will bind, provided that sufficient conditions are provided for binding to occur. For example, the guide RNA recognition sequence includes a sequence designed to be complementary to the guide RNA, wherein hybridization between the guide RNA recognition sequence and the DNA targeting sequence promotes formation of the CRISPR complex. Complete complementarity is not necessarily required, but sufficient complementarity must exist to induce hybridization and promote the formation of the CRISPR complex. The guide RNA recognition sequence also contains a cleavage site for the Cas protein, described in more detail below. The guide RNA recognition sequence may comprise any polynucleotide that can be located, for example, in the nucleus or cytoplasm of the cell or within an organelle of the cell, such as the mitochondrion or chloroplast.

The guide RNA recognition sequence in the target DNA can be targeted by (i.e., can be bound to, hybridize to, or be complementary to) a Cas protein or a gRNA. Suitable DNA/RNA binding conditions include physiological conditions normally present in cells. Other suitable DNA/RNA binding conditions are known.

Cas proteins can cleave nucleic acids at sites within or outside the nucleic acid sequence present in the target DNA to which the DNA-targeting segment of the gRNA will bind. A “cleavage site” includes the location in the nucleic acid at which the Cas protein creates a single-strand break or a double-strand break. For example, the formation of a CRISPR complex (comprising a gRNA that hybridizes to a guide RNA recognition sequence and is in complex with a Cas protein) involves placing (at or near) a nucleic acid sequence present in the target DNA to which the DNA-targeting segment of the gRNA will bind. cleavage of one or both strands (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the nucleic acid sequence) . The cleavage site may be on only one strand or on both strands of the nucleic acid. The cleavage site may be at the same location on both strands of the nucleic acid (creating blunt ends) or may be at different sites on each end (creating staggered ends (i.e., overhangs)). In some embodiments, the guide RNA recognition sequence of the nickase on the first strand is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, and the guide RNA recognition sequence of the nickase on the second strand. are separated by at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 250, at least 500, or at least 1,000 base pairs.

Site-specific cleavage of target DNA by Cas proteins occurs at positions determined by both i) the base pairing complementarity between the gRNA and the target DNA and ii) a short motif in the target DNA, called the protospacer adjacent motif (PAM). It can happen. The PAM may be flanked by a guide RNA recognition sequence. In some embodiments, the guide RNA recognition sequence may be flanked on the 3' end by a PAM. Alternatively, the guide RNA recognition sequence can be flanked on the 5' end by a PAM. For example, the cleavage site of the Cas protein may be about 1 to about 10 or about 2 to about 5 base pairs (e.g., 3 base pairs) upstream or downstream of the PAM sequence. In some cases (e.g., when Cas9 from S. pyogenes or a closely related Cas9 is used), the PAM sequence of the non-complementary strand may be 5'-N ₁ GG-3', where N ₁ is any DNA nucleotide and is immediately 3' of the guide RNA recognition sequence of the non-complementary strand of the target DNA. As such, the PAM sequence of the complementary strand will be 5'-CCN ₂ -3', where N ₂ is any DNA nucleotide and is immediately 5' of the guide RNA recognition sequence of the complementary strand of the target DNA. In some such cases, N ₁ and N ₂ may be complementary and the N ₁ -N ₂ base pair may be any base pair (e.g., N ₁ =C, N ₂ =G; N ₁ =G) , N ₂ =C; N ₁ =A and N ₂ =T; or N ₁ =T and N ₂ =A). For Cas9 from S. aureus, the PAM can be NNGRRT (SEQ ID NO: 13) or NNGRR (SEQ ID NO: 14), where N can be A, G, C, or T, and R can be G or It could be A. In some cases (e.g., for FnCpf1), the PAM sequence may be present upstream of the 5' end and has the sequence 5'-TTN-3'.

Examples of guide RNA recognition sequences include or include a DNA sequence complementary to the DNA-targeting segment of the gRNA in addition to the PAM sequence. For example, the target motif sequence can be a 20-nucleotide DNA sequence immediately preceding the NGG motif recognized by the Cas9 protein, such as GN ₁₉ NGG (SEQ ID NO: 15) or N ₂₀ NGG (SEQ ID NO: 16) (e.g. For example, see PCT Publication No. WO 2014/165825). Guanine at the 5' end can facilitate transcription by RNA polymerase in cells. Another example of a guide RNA recognition sequence may include two guanine nucleotides (e.g., GGN ₂₀ NGG; SEQ ID NO: 17) at the 5' end to facilitate efficient transcription by T7 polymerase in vitro ( See, for example, PCT Publication No. WO 2014/065596). Other guide RNA recognition sequences can be from about 4 to about 22 nucleotides in length, including a 5' G or GG and a 3' GG or NGG. In some embodiments, the other guide RNA recognition sequence may be about 14 to about 20 nucleotides in length.

The guide RNA recognition sequence can be any nucleic acid sequence that is endogenous or exogenous to the cell. The guide RNA recognition sequence may be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory sequence), or may include both.

In some embodiments, the guide RNA recognition sequence may be within the region corresponding to exon 5 of SEQ ID NO:1. In some embodiments, the guide RNA recognition sequence may comprise or is adjacent to positions 53575-53577 of SEQ ID NO:1. For example, the guide RNA recognition sequence includes positions corresponding to positions 53575 to 53577 of SEQ ID NO: 1, or about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45 thereof. , about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides. In some embodiments, the guide RNA recognition sequence may comprise or be adjacent to the start codon of the endogenous B4GALT1 gene or the stop codon of the endogenous B4GALT1 gene. For example, the guide RNA recognition sequence is within about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides of the start codon or stop codon. It can exist.

The methods and compositions disclosed herein utilize an exogenous donor sequence (e.g., a targeting vector or repair template) to cleave the endogenous B4GALT1 gene, either without cleavage of the endogenous B4GALT1 gene or following cleavage of the endogenous B4GALT1 gene with a nuclease agent . can be transformed. An exogenous donor sequence refers to any nucleic acid or vector that contains the necessary elements to enable site-specific recombination with the target sequence. The use of exogenous donor sequences in combination with nuclease agents can result in more precise modifications within the endogenous B4GALT1 gene by promoting homologous direct repair.

In this method, a nuclease agent cleaves the endogenous B4GALT1 gene to create a single-strand break (nick) or double-strand break, and the exogenous donor sequence is homologous or via non-homologous end joining (NHEJ)-mediated ligation. It recombines with the endogenous B4GALT1 gene through a direct repair event. Repair using an exogenous donor sequence removes or destroys the nuclease cleavage site, so the allele cannot be retargeted by nuclease agents.

The exogenous donor sequence comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may be single or double stranded and may exist in linear or circular form. For example, the exogenous donor sequence can be a single-stranded oligodeoxynucleotide (ssODN). Exemplary exogenous donor sequences are about 50 nucleotides to about 5 kb in length, about 50 nucleotides to about 3 kb in length, or about 50 to about 1,000 nucleotides in length. Other exemplary exogenous donor sequences are about 40 to about 200 nucleotides in length. For example, the exogenous donor sequence may be about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150, about 150 to about 160, about 160 to about 170, about 170 to about 180, about 180 to about 190 or about 190 to about 200 nucleotides in length. You can. Alternatively, the exogenous donor sequence may be about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, It may be about 700 to about 800, about 800 to about 900, or about 900 to about 1,000 nucleotides in length. Alternatively, the exogenous donor sequence is about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, about 2 kb to about 2.5 kb, about 2.5 kb to about 3 kb, about 3 kb to about 3.5 kb, about 3.5 kb to about 4 kb, It may be about 4 kb to about 4.5 kb or about 4.5 kb to about 5 kb in length. Alternatively, the exogenous donor sequence may be, for example, less than about 5 kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, less than about 2.5 kb, less than about 2 kb, less than about 1.5 kb, less than about 1 kb. Hereinafter, about 900 nucleotides or less, about 800 nucleotides or less, about 700 nucleotides or less, about 600 nucleotides or less, about 500 nucleotides or less, about 400 nucleotides or less, about 300 nucleotides or less. , may be up to about 200 nucleotides, up to about 100 nucleotides, or up to about 50 nucleotides in length.

In some embodiments, the exogenous donor sequence is an ssODN that is about 80 nucleotides to about 200 nucleotides long (e.g., about 120 nucleotides long). In another embodiment, the exogenous donor sequence is an ssODN that is about 80 nucleotides to about 3 kb in length. Such ssODNs may have homology arms, for example, each about 40 nucleotides to about 60 nucleotides long. Such ssODNs may also have homology arms, for example, each about 30 nucleotides to about 100 nucleotides long. The homology arms may be symmetrical (e.g., about 40 nucleotides each or about 60 nucleotides long each), or they may be asymmetric (e.g., one homology arm about 36 nucleotides long and about 60 nucleotides long each). one homology arm that is 91 nucleotides long).

The exogenous donor sequence may include modifications or sequences that provide additional desirable characteristics (e.g., modified or controlled stability; tracking or detection with a fluorescent label; binding site for a protein or protein complex, etc.). The exogenous donor sequence may include one or more fluorescent labels, purification tags, epitope tags, or combinations thereof. For example, the exogenous donor sequence may comprise one or more fluorescent labels (e.g., a fluorescent protein or other fluorophore or dye), such as at least 1, at least 2, at least 3, at least 4, or at least 5 fluorescent labels. there is. Exemplary fluorescent labels include fluorophores such as fluorescein (e.g., 6-carboxyfluorescein (6-FAM)), Texas Red, HEX, Cy3, Cy5, Cy5.5, Pacific Blue, 5-( and -6) -carboxytetramethylrhodamine (TAMRA) and Cy7. A wide range of fluorescent dyes are commercially available for labeling oligonucleotides (e.g., Integrated DNA Technologies). Such a fluorescent label (e.g., an internal fluorescent label) can be used to detect, for example, an exogenous donor sequence integrated directly into a truncated endogenous B4GALT1 gene with a protruding end that is compatible with the end of the exogenous donor sequence. The label or tag may be present at the 5' end, at the 3' end, or within the exogenous donor sequence. For example, an exogenous donor sequence can be conjugated at the 5' ^end with the IR700 fluorophore (5'IRDYE® 700) from Integrated DNA Technologies.

The exogenous donor sequence may also include a nucleic acid insert comprising a DNA segment to be integrated into the endogenous B4GALT1 gene. Integration of a nucleic acid insert within the endogenous B4GALT1 gene results in addition of the nucleic acid sequence of interest within the endogenous B4GALT1 gene, deletion of the nucleic acid sequence of interest within the endogenous B4GALT1 gene, or replacement (i.e. deletion and insertion) of the nucleic acid sequence of interest within the endogenous B4GALT1 gene. You can. Some exogenous donor sequences are designed for insertion of a nucleic acid insert within the endogenous B4GALT1 gene without any corresponding deletion within the endogenous B4GALT1 gene. Other exogenous donor sequences are designed to delete the nucleic acid sequence of interest within the endogenous B4GALT1 gene without any corresponding insertion of the nucleic acid insert. Another exogenous donor sequence is designed to delete the nucleic acid sequence of interest within the endogenous B4GALT1 gene and replace it with a nucleic acid insert.

The nucleic acid insert and corresponding nucleic acid within the endogenous B4GALT1 gene to be deleted and/or replaced can be of various lengths. Exemplary nucleic acid inserts or corresponding nucleic acids within the endogenous B4GALT1 gene to be deleted and/or replaced are from about 1 nucleotide to about 5 kb in length or from about 1 nucleotide to about 1,000 nucleotides in length. For example, the nucleic acid inserts and corresponding nucleic acids within the endogenous B4GALT1 gene to be deleted and/or replaced may have about 1 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50. , about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about It may be 130 to about 140, about 140 to about 150, about 150 to about 160, about 160 to about 170, about 170 to about 180, about 180 to about 190, or about 190 to about 200 nucleotides in length. Likewise, the nucleic acid inserts and corresponding nucleic acids within the endogenous B4GALT1 gene to be deleted and/or replaced are about 1 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about It may be 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, or about 900 to about 1,000 nucleotides in length. Likewise, the nucleic acid insert and corresponding nucleic acid within the endogenous B4GALT1 gene to be deleted and/or replaced range from about 1 kb to about 1.5 kb, from about 1.5 kb to about 2 kb, from about 2 kb to about 2.5 kb, from about 2.5 kb to about 3 kb, from about 3 kb to about 3 kb. It may be about 3.5 kb, about 3.5 kb to about 4 kb, about 4 kb to about 4.5 kb, or about 4.5 kb to about 5 kb in length.

Nucleic acid inserts may include genomic DNA or any other type of DNA. For example, a nucleic acid insert may include cDNA.

The nucleic acid insert may comprise a sequence homologous to all or part of the endogenous B4GALT1 gene (e.g., a portion of the gene encoding a specific motif or region of a B4GALT1 polypeptide). For example, the nucleic acid insert may contain one or more point mutations (e.g., 1, 2, 3, 4, 5 or more) or one or more nucleotide insertions or deletions compared to the sequence targeted for replacement in the endogenous B4GALT1 gene. It may include a sequence containing a.

The nucleic acid insert and corresponding nucleic acid within the endogenous B4GALT1 gene to be deleted and/or replaced include coding regions, such as exons; Non-coding regions, such as introns, untranslated regions, or regulatory regions (e.g., promoters, enhancers, or transcriptional repressor-binding elements); Or it may be any combination thereof.

The nucleic acid insert may also include a polynucleotide encoding a selectable marker. Alternatively, the nucleic acid insert may also lack a polynucleotide encoding a selectable marker. A selection marker may be contained within a selection cassette. In some embodiments, the selection cassette may be a self-deletion cassette. By way of example, a self-deletion cassette may comprise the Cre gene operably linked to the mouse Prm1 promoter (comprising two exons encoding the Cre recombinase separated by an intron) and the neomycin resistance gene operably linked to the human ubiquitin promoter. may include. Exemplary selection markers include neomycin phosphotransferase (neo ^r ), hygromycin B phosphotransferase (hyg ^r ), puromycin-N-acetyltransferase (puro ^r ), blasticidin S deaminase (bsr ^r ), xanthine/guanine phosphoribosyl transferase (gpt), or herpes simplex virus thymidine kinase (HSV-k), or combinations thereof. A polynucleotide encoding a selectable marker can be operably linked to a promoter that is active in the cell to be targeted. Examples of promoters are described elsewhere herein.

Nucleic acid inserts may also include reporter genes. Exemplary reporter genes include luciferase, β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and enhanced yellow fluorescent protein (eYFP). ), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean , which encodes T-Sapphire and alkaline phosphatase. Such reporter genes can be operably linked to a promoter that is active in the cell to be targeted. Examples of promoters are described elsewhere herein.

A nucleic acid insert may also include one or more expression cassettes or deletion cassettes. A particular cassette may contain one or more of a nucleotide sequence of interest, a polynucleotide encoding a selectable marker, and a reporter gene, along with various regulatory elements that affect expression. Examples of selectable markers and reporter genes that may be included are discussed in more detail elsewhere herein.

Nucleic acid inserts may include nucleic acids flanked by site-specific recombination recognition sequences. Alternatively, the nucleic acid insert may include one or more site-specific recombination recognition sequences. Although the entire nucleic acid insert may be flanked by such site-specific recombination recognition sequences, individual polynucleotides or any region of interest within the nucleic acid insert may also be flanked by such sites. Site-specific recombination recognition sequences capable of flanking the nucleic acid insert or any polynucleotide of interest in the nucleic acid insert include, for example, loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, It may include attp, att, FRT, rox, or combinations thereof. In some embodiments, the site-specific recombination site flanks a polynucleotide encoding a selectable marker and/or reporter gene contained within the nucleic acid insert. After integration of the nucleic acid insert into the endogenous B4GALT1 gene, sequences between site-specific recombination sites can be removed. In some embodiments, two exogenous donor sequences can be used, each having a nucleic acid insert containing a site-specific recombination site. Exogenous donor sequences can be targeted to the 5' and 3' regions flanking the nucleic acid of interest. After integration of the two nucleic acid inserts within the target genomic locus, the nucleic acid of interest between the two inserted site-specific recombination sites can be removed.

The nucleic acid insert may also include one or more restriction sites for restriction endonucleases (i.e., restriction enzymes), including type I, type II, type III, and type IV endonucleases. Type I and type III restriction endonucleases recognize specific recognition sequences, but typically cleave at variable positions from the nuclease binding site, which can be as much as hundreds of base pairs away from the cleavage site (recognition sequence). . In type II systems, restriction activity is independent of any methylase activity, and cleavage typically occurs at a specific site within or near the binding site. Most type II enzymes cleave palindromic sequences, but type IIa enzymes recognize non-palindromic recognition sequences and cleave outside the recognition sequence, and type IIb enzymes cleave the sequence twice, both outside the recognition sequence. Type II enzymes recognize asymmetric recognition sequences and cleave on one side at a defined distance of about 1 to 20 nucleotides from the recognition sequence. Type IV restriction enzymes target methylated DNA.

In some embodiments, the exogenous donor sequence comprises a 5' end complementary to one or more overhangs created by nuclease-mediated or Cas-protein-mediated cleavage at the target genomic locus (e.g., within the B4GALT1 gene) and /or has a short single-stranded region at the 3' end. These overhangs may also be referred to as 5' and 3' homology arms. For example, some exogenous donor sequences have 5' and/or 3' ends complementary to one or more overhangs created by Cas-protein-mediated cleavage in the 5' and/or 3' target sequence at the target genomic locus. has a short single-stranded region. In some embodiments, this exogenous donor sequence has a complementary strand only at the 5' end or only at the 3' end. For example, some of these exogenous donor sequences have a region of complementarity only at the 5' end, which is complementary to an overhang generated from the 5' target sequence at the target genomic locus, or at the 3' end complementary to an overhang generated from the 3' target sequence at the target genomic locus. ' It has a complementary region only at the end. Other such exogenous donor sequences have regions of complementarity at both the 5' and 3' ends. For example, other such exogenous donor sequences may be complementary at both the 5' and 3' ends, e.g., regions complementary to the first and second overhangs, respectively, generated by Cas-mediated cleavage at the target genomic locus. has For example, if the exogenous donor sequence is double stranded, the single-stranded complementarity region extends from the 5' end of the upper strand of the donor sequence and from the 5' end of the lower strand of the donor sequence, leaving a 5' overhang at each end. can be created. Alternatively, the single-stranded region of complementarity may extend from the 3' end of the upper strand of the donor sequence and from the 3' end of the lower strand of the template, creating a 3' overhang.

The region of complementarity can be of any length sufficient to facilitate ligation between the exogenous donor sequence and the endogenous B4GALT1 gene. Exemplary regions of complementarity are about 1 to about 5 nucleotides long, about 1 to about 25 nucleotides long, or about 5 to about 150 nucleotides long. For example, the region of complementarity may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or at least about 11. , at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least It may be about 24 or at least about 25 nucleotides long. Alternatively, the region of complementarity may be about 5 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150 nucleotides in length. It can be longer.

This region of complementarity may be complementary to the overhang created by the two pairs of nickases. Two double strand breaks having staggered ends, a first nickase and a second nickase cleaving opposing strands of DNA to produce a first double strand break, and cleaving opposing strands of DNA to produce a second double strand break. It can be produced by using a third nickase and a fourth nickase. For example, a Cas protein can be used to nick the first, second, third, and fourth guide RNAs and the corresponding first, second, third, and fourth guide RNAs. The first guide RNA and the second guide RNA recognition sequence are configured to create a first cleavage site such that nicks created by the first nickase and the second nickase on the first and second strands of the DNA produce a double strand break. (i.e., the first cleavage site includes nicks within the first and second guide RNA recognition sequences). Likewise, the third guide RNA and fourth guide RNA recognition sequences define a second cleavage site such that the nicks created by the third and fourth nickases on the first and second strands of DNA produce double-strand breaks. (i.e., the second cleavage site includes nicks in the third and fourth guide RNA recognition sequences). In some embodiments, the nick within the first guide RNA recognition sequence and the second guide RNA recognition sequence and/or the third guide RNA recognition sequence and the fourth guide RNA recognition sequence is an off-set nick that creates an overhang. ) can be. The offset window may be, for example, at least about 5 bp, at least about 10 bp, at least about 20 bp, at least about 30 bp, at least about 40 bp, at least about 50 bp, at least about 60 bp, at least about 70 bp, at least about 80 bp, at least about 90 bp, at least about It may be 100bp or more. In this embodiment, the double-stranded exogenous donor sequence can be designed with a single-stranded region of complementarity that is complementary to the overhang created by the nick in the first and second guide RNA recognition sequences and the nick in the third and fourth guide RNA recognition sequences. there is. This exogenous donor sequence can then be inserted by non-homologous end joining-mediated ligation.

In some embodiments, the exogenous donor sequence (i.e., targeting vector) includes a homology arm. If the exogenous donor sequence also includes a nucleic acid insert, the homology arms may flank the nucleic acid insert. For ease of reference, the homology arms are referred to herein as the 5' and 3' (i.e., upstream and downstream) homology arms. These terms relate to the relative position of the homology arm to the nucleic acid insert within the exogenous donor sequence.

The homology arm and target sequence correspond to each other if the two regions share a sufficient level of sequence identity to each other to serve as a substrate for a homologous recombination reaction. The sequence identity between a particular target sequence found in the exogenous donor sequence and the corresponding homology arm can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of sequence identity shared by the homology arm of the exogenous donor sequence (or fragment thereof) and the target sequence (or fragment thereof) is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%. %, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, may be at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, such that the sequences are susceptible to homologous recombination. You can experience it. Additionally, the corresponding region of homology between the homology arm and the corresponding target sequence can be of any length sufficient to promote homologous recombination. Exemplary homology arms are about 25 nucleotides to about 2.5 kb in length, about 25 nucleotides to about 1.5 kb in length, or about 25 to about 500 nucleotides in length. For example, a given homology arm (or each homology arm) and/or the corresponding target sequence may have about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 60. About 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 300 to about 350 , may comprise a corresponding homology region that is about 350 to about 400, about 400 to about 450, or about 450 to about 500 nucleotides in length, so that the homology arm undergoes homologous recombination with the corresponding target sequence within the endogenous B4GALT1 gene. It has sufficient homology to be experienced. Alternatively, a particular homology arm (or each homology arm) and/or the corresponding target sequence is about 0.5 kb to about 1 kb, about 1 kb to about 1.5 kb, about 1.5 kb to about 2 kb, or about 2 kb to about 2.5 kb. It may contain a corresponding homology region that is kb long. For example, the homology arms may each be about 750 nucleotides long. The homology arms may be symmetrical (each approximately the same amount of length) or asymmetrical (one longer than the other).

The homology arm may correspond to a locus that is native to the cell (e.g., the targeted locus). Alternatively, for example, it may correspond to a region of a heterologous or exogenous segment of DNA integrated into the genome of a cell, including, for example, a transgene, an expression cassette or a heterologous or exogenous region of DNA. In some embodiments, the homology arm of the targeting vector is a region of a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a human artificial chromosome, or any other contained in a suitable host cell. It may correspond to the manipulated area. In some embodiments, the homology arm of the targeting vector corresponds to or may be derived from a region of a BAC library, a cosmid library, or a P1 phage library, or may be derived from synthetic DNA.

When a nuclease agent is used in combination with an exogenous donor sequence, the 5' and 3' target sequences typically have a single-strand break (nick) or double-strand break at the nuclease cleavage site between the target sequence and the homology arm. It is located sufficiently adjacent to the nuclease cleavage site to facilitate homologous recombination events occurring. Nuclease cleavage sites include DNA sequences in which a nick or double-strand break is created by a nuclease agent (e.g., the Cas9 protein in complex with a guide RNA). The target sequence in the endogenous B4GALT1 gene corresponding to the 5' and 3' homology arms of the exogenous donor sequence is at a distance from the 5' and 3' target sequence, e.g., upon a single-strand break or double-strand break at a nuclease cleavage site. A nuclease cleavage site is "sufficiently located" if the distance between the homologous arms facilitates the occurrence of homologous recombination events. Thus, the target sequence corresponding to the 5' and/or 3' homology arms of the exogenous donor sequence is, for example, within at least 1 nucleotide of a given nuclease cleavage site, or at least 10 nucleotides from a given nuclease cleavage site. It can be anywhere from 1 nucleotide to about 1,000 nucleotides. In some embodiments, the nuclease cleavage site may be immediately adjacent to at least one or both target sequences.

The spatial relationship of the exogenous donor sequence and the target sequence corresponding to the homology arm of the nuclease cleavage site may vary. In some embodiments, the target sequence may be located 5' to a nuclease cleavage site, the target sequence may be located 3' to a nuclease cleavage site, or the target sequence may be located 3' to a nuclease cleavage site. can be adjacent to.

The present disclosure also provides methods of treatment and methods of treatment or prevention of cardiovascular conditions in a subject having the disease or at risk of having the disease using the methods disclosed herein to modify or alter the expression of the endogenous B4GALT1 gene. do. The present disclosure also provides methods for reducing expression of endogenous B4GALT1 mRNA, or providing a subject with a recombinant nucleic acid encoding a B4GALT1 polypeptide, providing an mRNA encoding a B4GALT1 polypeptide, or providing a B4GALT1 polypeptide. Provides a method of treating a cardiovascular condition in a subject having the disease or at risk of having the disease using the method and a method of treating or preventing the same. The methods may include introducing one or more nucleic acid molecules or proteins into a subject, into an organ of the subject, or into a cell of the subject (e.g., in vivo or in vitro).

In some embodiments, the present disclosure provides an mRNA encoding a B4GALT1 polypeptide (e.g., a polynucleotide as discussed herein, e.g., an mRNA comprising the sequence of SEQ ID NO: 4) for use in therapy. provides. In some such embodiments, the therapy is the treatment or prevention of a cardiovascular condition.

In some embodiments, the disclosure provides a B4GALT1 polypeptide (e.g., a polypeptide as discussed herein, e.g., a polypeptide comprising the sequence of SEQ ID NO: 8) for use in therapy. . In some such embodiments, the therapy is the treatment or prevention of a cardiovascular condition.

Subjects include humans and other mammalian subjects (e.g., felines, canines, rodents, mice, or rats) or non-mammalian subjects (e.g., poultry) receiving prophylactic or therapeutic treatment. Such a subject may be, for example, a subject (e.g., a human) who is not a carrier of variant B4GALT1 (or is only a heterozygous carrier of variant B4GALT1 ) and has or is susceptible to developing a cardiovascular condition.

Non-limiting examples of cardiovascular conditions include increased levels of one or more serum lipids. Serum lipids include cholesterol, LDL, HDL, triglycerides, HDL-cholesterol, and non-HDL cholesterol or their subclasses (e.g., HDL2, HDL2a, HDL2b, HDL2c, HDL3, HDL3a, HDL3b, HDL3c, HDL3d, LDL1). , LDL2, LDL3, lipoprotein A, Lpa1, Lpa1, Lpa3, Lpa4 or Lpa5). Cardiovascular conditions may include increased levels of coronary artery calcification. Cardiovascular conditions may involve type IId glycosylation (CDG-IId). Cardiovascular conditions may include increased levels of fat around the heart. Cardiovascular conditions may include atherothrombotic conditions. Atherothrombotic conditions may involve increased levels of fibrinogen. Atherothrombotic conditions may include fibrinogen-mediated blood clots. Cardiovascular conditions may involve increased levels of fibrinogen. Cardiovascular conditions may include fibrinogen-mediated blood clots. Cardiovascular conditions may include blood clots that form from involvement of fibrinogen activity. Fibrinogen-mediated blood clots, or blood clots formed from involvement of fibrinogen activity, can be present within any vein or artery within the body.

These methods may include genome editing or gene therapy. For example, an endogenous B4GALT1 gene that is not variant B4GALT1 can be modified to include a mutation associated with variant B4GALT1 (i.e., replacement of asparagine with serine at the position corresponding to position 352 of the full-length/mature B4GALT1 polypeptide). As another example, the endogenous B4GALT1 gene, but not the endogenous B4GALT1 variant, can be knocked out or inactivated. Likewise, an endogenous B4GALT1 gene other than variant B4GALT1 can be knocked out or inactivated, and a B4GALT1 gene containing a variant associated with variant B4GALT1 (e.g., a full variant B4GALT1 or a minigene containing the variant) can be introduced and expressed. there is. Similarly, the endogenous B4GALT1 gene, but not variant B4GALT1 , can be knocked out or inactivated, recombinant DNA encoding the B4GALT1 variant polypeptide can be introduced and expressed, and mRNA encoding the B4GALT1 variant polypeptide can be introduced and expressed. (e.g., intracellular protein replacement therapy) and/or variant B4GALT1 polypeptides may be introduced (e.g., protein replacement therapy).

In some embodiments, the methods include introducing and expressing a recombinant B4GALT1 gene comprising a modification associated with a B4GALT1 rs551564683 variant (e.g., a full variant B4GALT1 or a minigene comprising the modification), or a variant B4GALT1 polypeptide or fragment thereof. Introducing and expressing a recombinant nucleic acid (e.g., DNA) encoding, or introducing and expressing one or more mRNAs encoding a variant B4GALT1 polypeptide or fragment thereof (e.g., intracellular protein replacement therapy), or introducing a variant B4GALT1 polypeptide or fragment thereof (e.g., protein replacement therapy) without knocking out or inactivating an endogenous B4GALT1 gene other than variant B4GALT1 . In some embodiments, these methods also include variants other than B4GALT1 . This can be performed in combination with methods in which endogenous B4GALT1 mRNA is targeted for reduced expression, such as through the use of antisense RNA, siRNA or shRNA.

DNA encoding the B4GALT1 gene or minigene or variant B4GALT1 polypeptide or fragment thereof can be introduced and expressed in the form of an expression vector that does not modify the genome, or it can be targeted to be genomically integrated into the endogenous B4GALT1 locus. or it may be introduced to be genomically integrated within a locus other than the endogenous B4GALT1 locus, such as a safe harbor locus. The genomically integrated B4GALT1 gene may be operably linked to the B4GALT1 promoter or another promoter, such as an endogenous promoter, at the site of integration. A safe harbor locus is a chromosomal region where a transgene can be stably and feasibly expressed in all loci of interest without adversely affecting gene structure or expression. A safe harbor locus may have one or more or all of the following features, for example: (1) a distance greater than about 50 kb from the 5' end of any gene; A distance greater than about 300 kb from any cancer-related gene; A distance greater than about 300 kb from any microRNA; outside the gene transcription unit; and outside the super-conservation region. Examples of suitable safe harbor loci include, but are not limited to, the adeno-associated viral site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) gene locus, and the human ortholog of the mouse ROSA26 locus.

In some embodiments, the methods include a method of treating a subject who is not a carrier of variant B4GALT1 (or is only a heterozygous carrier of variant B4GALT1) and has or is susceptible to developing a cardiovascular condition, comprising: In the cell of: a) a nuclease agent (or nucleic acid encoding) that binds to a nuclease recognition sequence in the endogenous B4GALT1 gene, wherein the nuclease recognition sequence comprises or corresponds to positions 53575 to 53577 of SEQ ID NO: 1 adjacent); and b) a 5' homology arm that hybridizes to the target sequence 5' at positions 53575 to 53577 of SEQ ID NO: 1, and a nucleic acid sequence encoding a serine flanked by the 5' homology arm and the 3' homology arm. and introducing an exogenous donor sequence comprising a nucleic acid insert. The nuclease agent can cleave the endogenous B4GALT1 gene within a cell in a subject, wherein the exogenous donor sequence can recombine with the endogenous B4GALT1 gene within the cell, wherein upon recombination of the exogenous donor sequence with the endogenous B4GALT1 gene, serine The coding nucleic acid sequence is inserted at nucleotides corresponding to positions 53575 to 53577 of SEQ ID NO: 1. Examples of nuclease agents (e.g., Cas9 protein and guide RNA) that can be used in these methods are disclosed elsewhere herein.

In some embodiments, the methods include a method of treating a subject who is not a carrier of variant B4GALT1 (or is only a heterozygous carrier of variant B4GALT1) and has or is susceptible to developing a cardiovascular condition, comprising: A 5' homology arm that hybridizes to the target sequence 5' at a position corresponding to positions 53575 to 53577 of SEQ ID NO: 1, and a 3' homology arm that hybridizes to the target sequence 3' of positions 53575 to 53577 of SEQ ID NO: 1. introducing an exogenous donor sequence comprising an arm and a nucleic acid insert comprising a nucleotide sequence encoding a serine flanked by a 5' homology arm and a 3' homology arm. The exogenous donor sequence can be recombined with the endogenous B4GALT1 gene in a cell, wherein, upon recombination of the exogenous donor sequence with the endogenous B4GALT1 gene, the nucleotide sequence encoding serine is in the nucleotides corresponding to positions 53575 to 53577 of SEQ ID NO: 1. is inserted.

Some such methods include methods of treating a subject who is not a carrier of variant B4GALT1 ant (or is only a heterozygous carrier of variant B4GALT1 ) and has or is susceptible to developing a cardiovascular condition, comprising: Into: a) introducing a nuclease agent (or encoding nucleic acid) that binds to a nuclease recognition sequence in the endogenous B4GALT1 gene, wherein the nuclease recognition sequence comprises a start codon for the endogenous B4GALT1 gene or or within about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides of the start codon, or is selected from SEQ ID NOs: 9 to 12. . The nuclease agent cleaves the endogenous B4GALT1 gene in cells within the subject and prevents its expression.

In some embodiments, the methods include a method of treating a subject who is not a carrier of variant B4GALT1 (or is only a heterozygous carrier of variant B4GALT1 ) and has or is susceptible to developing a cardiovascular condition, comprising: In a cell of: a) a nuclease agent (or encoding nucleic acid) that binds to a nuclease recognition sequence in the endogenous B4GALT1 gene, wherein the nuclease recognition sequence comprises the start codon for the endogenous B4GALT1 gene or is about the start codon within 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500 or about 1,000 nucleotides or selected from SEQ ID NOs: 9-12); and b) introducing an expression vector containing a recombinant B4GALT1 gene comprising a nucleotide sequence at positions 53575 to 53577 encoding a serine at a position corresponding to position 352 of the full-length/mature B4GALT1 polypeptide. The expression vector may not be integrated into the genome. Alternatively, a targeting vector (i.e., an exogenous donor sequence) can be introduced, which contains a nucleotide sequence at positions 53575 to 53577 that encodes a serine at position 352 corresponding to the full-length/mature B4GALT1 polypeptide. Contains the recombinant B4GALT1 gene. The nuclease agent can cleave the B4GALT1 gene in cells within the subject and prevent its expression, and the expression vector can express the recombinant B4GALT1 gene in cells within the subject. Alternatively, the recombinant B4GALT1 gene, integrated into the genome, can be expressed in cells within the subject. Examples of nuclease agents (e.g., nuclease active Cas9 protein and guide RNA) that can be used in these methods are disclosed elsewhere herein. Examples of suitable guide RNAs and guide RNA recognition sequences are also disclosed elsewhere herein. Step b) alternatively comprises a nucleic acid encoding a B4GALT1 polypeptide that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the variant B4GALT1 Asn352Ser polypeptide or fragment thereof. (e.g., DNA) and/or an expression vector comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the variant B4GALT1 mRNA or fragment thereof. Alternatively, it may include introducing a targeting vector. Likewise, step b) also encodes a B4GALT1 Asn352Ser polypeptide that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the variant B4GALT1 Asn352Ser polypeptide or fragment thereof; /or introducing an mRNA having complementary DNA (or a portion thereof) that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 % identical to the variant B4GALT1 mRNA or fragment thereof. It can be included. Likewise, step b) also comprises a protein comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the variant B4GALT1 Asn352Ser polypeptide or fragment thereof. This may include introducing

In some embodiments, the second nuclease agent is also introduced into the subject or into a cell of the subject, wherein the second nuclease agent binds to a nuclease recognition sequence within the endogenous B4GALT1 gene, and wherein the second nuclease agent binds to a nuclease recognition sequence within the endogenous B4GALT1 gene. The recognition sequence includes a second stop codon for the endogenous B4GALT1 gene or about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500 or about exists within 1,000 nucleotides or is selected from SEQ ID NOs: 9-12, wherein the nuclease agent cleaves the endogenous B4GALT1 gene in the cell within both the first nuclease recognition sequence and the second nuclease recognition sequence; wherein the cell is transformed to contain a deletion between a first nuclease recognition sequence and a second nuclease recognition sequence. In some embodiments, the second nuclease agent may be Cas9 protein and guide RNA. Suitable guide RNAs and guide RNA recognition sequences adjacent to stop codons are disclosed elsewhere herein.

In some embodiments, the methods may also include a method of treating a subject who is not a carrier of variant B4GALT1 (or is only a heterozygous carrier of variant B4GALT1 ) and has or is susceptible to developing a cardiovascular condition, which method comprises: or into a cell of the subject: introducing an antisense RNA, siRNA, or shRNA that hybridizes to a sequence within a region within the endogenous B4GALT1 mRNA. For example, antisense RNA, siRNA or shRNA can hybridize to a sequence within exon 5 of SEQ ID NO:3 ( B4GALT1 mRNA) and reduce the expression of B4GALT1 mRNA in cells within a subject. In some embodiments, these methods may further comprise introducing into the subject an expression vector comprising a recombinant B4GALT1 gene comprising a nucleotide sequence encoding a serine inserted at positions 53575 to 53577 of SEQ ID NO: 2. there is. The expression vector may not be integrated into the genome. Alternatively, a targeting vector (i.e., an exogenous donor sequence) can be introduced, which contains a recombinant B4GALT1 gene comprising a nucleic acid sequence encoding serine at positions corresponding to positions 53575 to 53577 of SEQ ID NO:2. In methods in which an expression vector is used, the expression vector is capable of expressing the recombinant B4GALT1 gene in cells within the subject. Alternatively, in methods where the recombinant B4GALT1 gene is integrated into the genome, the recombinant B4GALT1 gene can be expressed in cells within the subject.

In some embodiments, such methods comprise a method comprising a variant B4GALT1 polypeptide encoding a B4GALT1 polypeptide that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a variant B4GALT1 Asn352Ser polypeptide or fragment thereof. Expression comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a nucleic acid (e.g., DNA) and/or variant B4GALT1 mRNA or fragment thereof. It may include introducing a vector or targeting vector. Likewise, these methods alternatively encode a polypeptide that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the variant B4GALT1 Asn352Ser polypeptide or fragment thereof, and/ or introducing an mRNA having complementary DNA (or a portion thereof) that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 % identical to the variant B4GALT1 mRNA or fragment thereof. can do. Likewise, these methods may alternatively be used to produce a polypeptide comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the variant B4GALT1 Asn352Ser polypeptide or fragment thereof. It may include introducing .

In some embodiments, such methods may include methods of treating a subject who is not a carrier of variant B4GALT1 (or is only a heterozygous carrier of variant B4GALT1) and has or is susceptible to developing a cardiovascular condition, the method comprising: Introducing an expression vector within or into a cell of a subject, wherein the expression vector comprises a nucleotide sequence at positions 53575 to 53577 that encodes a serine at a position corresponding to position 352 of the full-length/mature B4GALT1 polypeptide. and a recombinant B4GALT1 gene, wherein the expression vector expresses the recombinant B4GALT1 gene in a cell within the subject. The expression vector may not be integrated into the genome. Alternatively, a targeting vector (i.e., an exogenous donor sequence) can be introduced, which contains nucleotides at positions 53575 to 53577 of SEQ ID NO: 2, which encodes a serine at a position corresponding to position 352 of the full-length/mature B4GALT1 polypeptide. Contains a recombinant B4GALT1 gene comprising the sequence. In methods in which an expression vector is used, the expression vector is capable of expressing the recombinant B4GALT1 gene in cells within the subject. Alternatively, in methods where the recombinant B4GALT1 gene is integrated into the genome, the recombinant B4GALT1 gene can be expressed in cells within the subject.

These methods alternatively include nucleic acids ( For example, DNA) and/or an expression vector comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the variant B4GALT1 mRNA or fragment thereof, or It may include introducing a targeting vector. Likewise, these methods alternatively encode a polypeptide that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the variant B4GALT1 polypeptide or fragment thereof. It may comprise introducing an mRNA having complementary DNA (or a portion thereof) that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 % identical to the variant B4GALT1 mRNA or fragment thereof. You can. Likewise, these methods may alternatively produce a protein comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 % identical to the variant B4GALT1 Asn352Ser polypeptide or fragment thereof. This may include introducing

Expression vectors and recombinant B4GALT1 genes suitable for use in any of the above methods are disclosed elsewhere herein. For example, the recombinant B4GALT1 gene can be a complete B4GALT1 variant gene or a B4GALT1 minigene in which one or more non-essential segments of the gene have been deleted relative to the corresponding wild-type B4GALT1 gene. By way of example, the deleted segment may include one or more intron sequences and the minigene may include exons 1-6. An example of a complete B4GALT1 variant gene is one that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:2.

In some embodiments, such methods include methods of modifying cells in a subject having or susceptible to a cardiovascular condition. In these methods, the nuclease agent and/or exogenous donor sequence and/or recombinant expression vector are used to delay the onset, reduce the severity, and/or increase the severity of at least one sign or symptom of the cardiovascular condition being treated. It can be introduced into the cell through administration in an effective regimen, meaning the dosage, route of administration and frequency of administration that inhibits and/or improves the exacerbation. The term “symptoms” refers to subjective evidence of disease as perceived by a subject, and “signs” refers to objective evidence of disease as observed by a physician. If the subject already suffers from the disease, the therapy may be referred to as a therapeutically effective therapy. If the subject has an increased risk for the disease compared to the general population but has not yet experienced symptoms, the therapy may be referred to as a prophylactically effective therapy. In some instances, therapeutic or prophylactic efficacy may be observed in an individual patient compared to historical controls or past experience in the same subject. In other examples, therapeutic or prophylactic efficacy can be demonstrated in preclinical or clinical trials in a population of treated subjects compared to a control population of untreated subjects.

Delivery may be by any suitable method, as disclosed elsewhere herein. For example, nuclease agents or exogenous donor sequences or recombinant expression vectors can be used, for example, in vector delivery, viral delivery, particle-mediated delivery, nanoparticle-mediated delivery, liposome-mediated delivery, exosome-mediated delivery, etc. Delivery may be by mediated delivery, lipid-mediated delivery, lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediated delivery, or implantable-device-mediated delivery. Specific examples include hydrodynamic delivery, virus-mediated delivery, and lipid-nanoparticle-mediated delivery.

Administration can be by any suitable route, including but not limited to, for example, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. It can be depended on. For example, a specific example often used for protein replacement therapy is intravenous infusion. The frequency and frequency of administration may depend on the half-life of the nuclease agent or exogenous donor sequence or recombinant expression vector, the condition of the subject, and the route of administration, among other factors. The pharmaceutical composition is preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions may be presented in unit dosage form (i.e., dosage for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The formulation will depend on the route of administration chosen. The term “pharmaceutically acceptable” means that the carrier, diluent, excipient or adjuvant is compatible with the other ingredients of the formulation and is not substantially harmful to its recipient.

These other methods include in vitro methods in cells from subjects with or susceptible to developing cardiovascular conditions. Cells with the targeted genetic modification can then be transplanted back into the subject.

The present disclosure provides methods of reducing LDL in a subject in need thereof by reducing expression of endogenous wild-type B4GALT1 or increasing expression of B4GALT1 Asn352Ser, by any of the methods described herein. . The present disclosure provides methods for reducing total cholesterol in a subject in need thereof by reducing expression of endogenous wild-type B4GALT1 or increasing expression of B4GALT1 Asn352Ser, by any of the methods described herein. to provide. The present disclosure provides methods of reducing fibrinogen in a subject in need thereof by reducing expression of endogenous wild-type B4GALT1 or increasing expression of B4GALT1 Asn352Ser, by any of the methods described herein. . The present disclosure provides methods of reducing eGFR in a subject in need thereof by reducing expression of endogenous wild-type B4GALT1 or increasing expression of B4GALT1 Asn352Ser, by any of the methods described herein. . The present disclosure provides for increasing AST, but not ALT, in a subject in need thereof by reducing expression of endogenous wild-type B4GALT1 or increasing expression of B4GALT1 Asn352Ser, by any of the methods described herein. Provides a way to increase The present disclosure provides methods of increasing creatinine in a subject in need thereof by reducing expression of endogenous wild-type B4GALT1 or increasing expression of B4GALT1 Asn352Ser, by any of the methods described herein. .

The disclosure also provides a method of diagnosing a risk of developing a cardiovascular condition or a method of diagnosing a risk of developing a cardiovascular condition and a method of treating a cardiovascular condition in a subject in need thereof, the method comprising: Requesting a test that provides analysis of a sample from the subject for the presence or absence of a variant B4GALT1 gene, mRNA, cDNA or polypeptide, as described herein; and, in a subject that does not have the variant B4GALT1 gene, mRNA, cDNA, or polypeptide, administering to the subject a therapeutic agent as described herein. Any of the tests described herein can be used to determine the presence or absence of a variant B4GALT1 gene, mRNA, cDNA, or polypeptide.

The present disclosure also provides for the use of a medicament to reduce LDL, reduce total cholesterol, reduce fibrinogen, reduce eGFR, increase AST (but not ALT), and increase creatinine in a subject in need thereof. Provided is the use of any of the variant B4GALT1 genes, mRNA, cDNA, polypeptides and hybridized nucleic acid molecules disclosed herein in manufacturing. The disclosure also provides for the use of any of the variant B4GALT1 genes, mRNA, cDNA, polypeptides and hybridized nucleic acid molecules in the manufacture of a medicament for the treatment of coronary artery disease, coronary artery calcification and related disorders.

The present disclosure also provides methods for reducing LDL, reducing total cholesterol, reducing fibrinogen, reducing eGFR, increasing AST (but not ALT), and increasing creatinine in a subject in need thereof. Provided are uses of any of the variant B4GALT1 genes, mRNA, cDNA, polypeptides and hybridized nucleic acid molecules disclosed in.

The present disclosure also provides use of any of the variant B4GALT1 genes, mRNA, cDNA, polypeptides and hybridized nucleic acid molecules for the treatment of coronary artery disease, coronary artery calcification, type IId glycosylation (CDG-IId) and related disorders. do.

The disclosure also provides the use of any of the variant B4GALT1 genes, mRNA, cDNA, polypeptides and hybridized nucleic acid molecules disclosed herein for modifying the B4GALT1 gene in a cell in a subject in need thereof.

The disclosure also provides for the use of any of the variant B4GALT1 genes, mRNA, cDNA, polypeptides and hybridized nucleic acid molecules disclosed herein for altering the expression of the B4GALT1 gene in a cell in a subject in need thereof.

The disclosure also provides for the use of any of the variant B4GALT1 genes, mRNA, cDNA, polypeptides, and hybridized nucleic acid molecules disclosed herein for diagnosing the risk of developing any of the cardiovascular conditions disclosed herein.

The disclosure also provides for the use of any of the variant B4GALT1 genes, mRNA, cDNA, polypeptides, and hybridized nucleic acid molecules disclosed herein for diagnosing a subject with any of the cardiovascular conditions disclosed herein.

All patent applications, websites, other publications, registration numbers, etc. cited above or hereinafter are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. . If different versions of a sequence are associated with registration numbers at different times, the version associated with the registration number at the effective filing date of this application is meaningful. Effective filing date means prior to the actual filing date or, if applicable, the filing date of the priority application to which the registration number refers. Likewise, if different versions of publications, websites, etc. are published at different times, this means the version published closest to the effective filing date of this application, unless otherwise indicated. Any feature, step, element, embodiment or aspect of the disclosure may be used in combination with any other unless specifically indicated otherwise. Although the invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Nucleotide and amino acid sequences referred to herein are presented using standard letter abbreviations for nucleotide bases and one-letter codes for amino acids. Nucleotide sequences follow the standard convention of starting at the 5' end of the sequence and proceeding toward the 3' end (i.e., left to right on each line). Although only one strand of each nucleotide sequence is shown, complementary strands are understood to be included by any reference to the indicated strand. Amino acid sequences follow the standard convention of starting at the amino terminus of the sequence and proceeding toward the carboxy terminus (i.e., left to right in each line).

U.S. Application No. 62/659,344 (filed April 18, 2018), U.S. Application No. 62/550,161 (filed August 25, 2017), and U.S. Patent No. 62/515,140 (filed June 5, 2017) ) are each incorporated herein by reference in their entirety.

The following examples are provided to describe the embodiments in more detail. It is intended to be illustrative, not limiting, of the claimed embodiments.

Example

Example 1: Determination of novel loci on chromosome 9p.21 associated with serum lipid traits at genome-wide statistical significance

Materials and Methods:

Chip genotyping and QC: Genomic DNA was extracted from whole blood from OOA individuals and quantified using picogreen. Whole-genome genotyping was performed using Affymetrix 500K and 6.0 chips at the University of Maryland Biopolymer Core Facility. The BRLMM algorithm was used for genotype calling. Samples with call rates below 0.93, high levels of Mendelian error, or gender mismatch were excluded. SNPs with Col ratio less than 0.95, HWE p-value less than 1.0E-6 or MAF less than 0.01 were excluded. SNPs on chromosomes X and Y and the mitochondrial genome were also excluded.

WGS and QC: Library preparation and whole genome sequencing were performed by the Broad Institute of MIT and Harvard. The NHLBI Informatics Resource Core at the University of Michigan performed alignment, base calling, and sequence quality scoring of all TOPMed samples, with read depths of at least 10 used in the analysis. Bcf files were delivered for all variants that passed all quality filters. Additional QC was applied to this file, including removing all regions on the LCR, or X chromosome. Variants with deletion rate greater than 5%, HWE p-value less than 1.0E-09 and MAF less than 0.1% were also removed. Sample QC was performed to remove samples with missing rates greater than 5%, high levels of Mendelian error (in some instances), or identical (MZ) pairs (one of each pair).

WES and QC: Exome capture and sequencing were performed at the Regeneron Genetics Center (RGC) as described in more detail below. Briefly, captured libraries were sequenced on an Illumina HiSeq 2500 platform with v4 chemistry using paired-end 75bp reads. Paired-end sequencing of captured bases was performed to ensure that more than 85% of the bases were covered by 20× or greater (sufficient to call heterozygous variants across most of the targeted bases). Read alignment and variant calling were performed using BWA-MEM and GATK as implemented in the RGC DNAseq analysis pipeline. Samples with Colby less than 0.90, high levels of Mendelian error, or identical (MZ) pairs (one of each pair) or gender mismatch were excluded. SNPs with Col ratios less than 0.90 and monomorphic SNPs were also excluded. SNPs on chromosomes X and Y and the mitochondrial genome were also excluded.

Correlation analysis: Fasting blood samples were collected and used for lipid analysis. LDL was calculated using the Friedewald formula, and in some analyzes using subjects taking lipid-lowering medications, LDL levels were adjusted by dividing by 0.7. Genetic linkage analysis was performed using linear mixed models to account for familial correlations using pedigree-based kinship matrices and/or family correction to estimate kinship from WES. Analyzes were also adjusted for age, age squared, gender, cohort, and APOB R3527Q genotype. APOB R3527Q is abundant in the Amish and has previously been identified as having a strong effect on LDL levels (58 mg/dl) (Shen et al., Arch Intern. Med., 2010, 170, 1850-1855), and was therefore found in the LDL analysis. The effects of these variants were considered. A genome-wide corrected p-value of 5.0E-08 was used as the significance threshold.

Identification of the association between chromosome 9p region and LDL using Genome Wide Association Study (GWAS):

To identify causative variants in novel genes associated with cardiovascular risk factors, genome-wide linkage analysis was performed using 1852 Old Order Amish subjects genotyped using Affymetrix 500K and 6.0 chips. Table 1 shows the basic characteristics of these participants.

Almost all (96%) of the WGS fine mapping samples were included in the GWAS discovery samples.

Only 30% of WES samples were included in GWAS or WGS samples.

As shown in Figure 1, we found a strong novel association signal between LDL and a locus on chromosome 9p. The predominant associated SNP was rs855453 (p=2.2E-08), with a frequency of 15% in the Amish and 25% in the general population. The minor 'T' allele was associated with lower LDL levels of 10 mg/dL. Therefore, this GWAS SNP is common in both Amish and non-Amish, has a large effect size, but was not identified in any of the large GWAS meta-analyses. These features are consistent with those of previous studies ( APOC3 and LIPE ) and, on this basis, suggest that these GWAS SNPs are not causal/functional variants in these regions but are associated with another variant that is rare in the general population but common in the Amish population (disequilibrium). It was concluded that it was linkage disequilibrium (LD). Additionally, multiple studies based on five independent crosses of multiple strains also found that the syntenin region of the rat genome, located on rat chromosome 5, harbors QTL for serum cholesterol and triglyceride levels (The Rat Genome Database (RGD). Scl12.26. 35. 44, 54 and Stl 28).

Confirmation using whole exome sequencing (WES):

We subsequently used high-quality QC'd WES on 4,565 Amish individuals whose basic characteristics are shown in Table 1. Results of a mixed model exome wide analysis of LDL identified the B4GALT1 rs551564683 missense variant as the most significant association with a p-value of 3.3E-18 and an effect size of lower LDL of 14.7 mg/dl. The rs551564683 variant had a MAF of 6% in the Amish, but was very rare in the general population. This variant is present in dbSNP without frequency or population information, is not present in the ExAC database (60,000 samples), and WGS from 15,387 Biamish in the NHLBI Trans-Omics for Precision Medicine (TOPMed) dataset. Only one copy was found in Additionally, only 79 heterozygotes and 5 homozygotes of this variant were found in a collective data set of different population cohorts available to researchers - a total of 125,401 individuals (over 1,000-fold more abundant in the Amish population). This missense variant is located 500 kb away from the GWAS variant with an r2 estimate of LD of 0.5. There is no perfectly related variant of rs551564683; In fact, the next most significant SNP is rs149557496 with a p-value of E-14. Therefore, the strength of the rs551564683 association confirms that the chromosome 9 GWAS locus is real and that rs551564683 has all the predicted characteristics of an episodic variant.

Precise mapping of the chromosome 9p region using whole genome sequencing (WGS):

Gaps in exome sequencing were filled using WGS, which was available for smaller samples, providing additional evidence that rs551564683 was causal. WGS data for 1083 OOA were generated as part of the TOPMed program. Table 1 shows the basic characteristics of WGS samples. WGS captures all SNPs and Indels (both coding and non-coding) that may be correlated with upstream variants within the region of interest. Because the top variant has a frequency of approximately 6%, it is unlikely that the variant caller will have insufficient sequence reads to cause missed variants. However, there may be variants excluded during the QC procedure. By examining variants that did not pass QC, two additional variants were added to the analysis. Linkage analysis identified the missense SNP (N352S) rs551564683 in the B4GALT1 gene as the variant most significantly associated with LDL in this region with a p-value of 2.9E-06 and an effect size of -16.4 mg/dl (see Table 2 ).

The TOPMed WGS data set provided 20 variants associated with LDL that were highly but not perfectly correlated with the top hit rs551564683 (r2 = 0.83-0.94), with a p-value of 2.9E-06 to 2.5E-05 ( (see red in Figure 2). Conditional analysis adjustment for rs551564683 completely abolished the association signal of 20 variants and did not reveal any other signal in this region, strongly suggesting a single causal variant.

Careful examination of these 20 variants (see red in Figure 2) divided the variants into two groups: 7 red variants in shaded triangles and 13 unshaded red variants. The seven red variants within the shaded triangle were almost completely correlated with each other, with the top hit rs551564683 and an r2 of 0.83. These 7 variants were safely excluded as causal/functional based on 3 reasons: 1) they are relatively common outside of OOA (maf > 1%), 2) they were reported within TOPMed in the Framingham Heart Study: 3) one of these seven variants had an LDL association of 6.3E-14 versus 3.3E-18 for the top hit rs551564683 in WES data of 4,565 OOA subjects; It had a p-value.

Another group of variants within the shaded squares in Figure 2 also had an association p-value of only about 10E-6, were almost completely correlated with each other, and had an r2 of 0.68 with the top hit rs551564683. This group was also excluded as causal/functional because it is relatively common outside of OOA (maf about 4%) and did not show any association with LDL in the 3877 samples from FHS in TOPMed.

In Figure 2, the top hit rs551564683 and 13 unshaded red variants (extending over 4 Mb from 31.5 Mb to 35.5 Mb on the short arm of chromosome 9) remained. As described above, these 13 variants were almost completely correlated with each other and had an r2 of 0.91 to 0.94 with the top hit rs551564683. Among these variants, the top hit rs551564683 was the only coding variant, which was classified as damaging and deleterious by 5 out of 9 algorithms predicting the effect of variants on protein function. The top hit rs551564683 and these 13 variants had a MAF of 6% in OOA, but are almost absent in the general population.

Haplotype analysis:

Incomplete r2 between distinct loci is the result of recombination events. A detailed analysis of the primary 14-SNP haplotype was performed. Figure 3 shows these three major haplotypes within this 4 Mb region. There were 115 subjects (1 homozygote and 114 heterozygotes) with haplotype A, which had identical genotypes at 14 SNPs, but did not provide any information about which SNP may be causal. . Six subjects had haplotype B, containing a heterozygous genotype at rs551564683 and four upstream SNPs, and seven subjects had haplotype C, containing a heterozygous genotype at rs551564683 and nine downstream SNPs. Recombinant haplotypes B and C clustered in related subjects, providing evidence that they are not an artifact of genotyping error. Table 3 shows the p-value of rs551564683 after adding individuals with haplotypes B and C to a single group compared to individuals with haplotype A.

Adding haplotypes B and C individually improved the p-value, and adding both improved the p-value even better. The improved p-value indicated that both haplotypes B and C carry the causal allele. The only SNP in common between B and C was rs551564683, which was considered to be a causal variant.

B4GALT1 congenital impairment of glycosylation supports a functional role for rs551564683:

A phenotype-wide association study (PheWAS) was performed to test the association of rs551564683 with all traits in the Amish database. The strongest association after LDL (p= 3.3E-18) and total cholesterol (p= 3.0E-18) was found for aspartate transaminase (AST) (p= 3.0E-8), where the minor allele Homozygotes had a two-fold increase in AST levels than wild-type homozygotes. Higher AST has already been reported in a case of congenital disorder of glycosylation (CGD) caused by a frameshift insertion in B4GALT1 resulting from a truncated dysfunctional protein. Moreover, a strong association with fibrinogen levels (p=5.0E-4) was observed, where homozygous levels were approximately 20% lower than wild type, consistent with blood coagulation defects in the same CDG patients. Moreover, in a small experiment, a 50% increase in creatine kinase serum levels (p=0.02) was found in 13 minor allele homozygotes compared to 13 wild-type homozygotes. This consistency in the phenotypes associated with the missense SNP and caused by the truncated insertion in B4GALT1 further supports the evidence that the B4GALT1 rs551564683 SNP is a causal/functional gene and variant in this region.

The association between lipid subclasses and rs551564683 was examined in 759 Amish individuals, and as shown in Table 4, associations with lower levels of almost all subclasses were observed with significant or non-significant p-values.

Coronary calcification score, aortic calcification score and pericardial fat showed a trend toward association with lower levels but did not have a significant p-value.

PheWAS also found that rs551564683 was associated with higher creatinine and lower eGFR, as well as higher hematocrit and lower basophils.

Example 2: Sample preparation and sequencing

Genomic DNA sample concentrates were obtained from Amish subjects, then transferred to in-house facilities and stored at -80°C (LiCONiC TubeStore) until sequence analysis. Sample quantity was determined by fluorescence (Life Technologies) and quality was assessed by running 100 ng of sample on a 2% pre-cast agarose gel (Life Technologies).

DNA samples were normalized and each sample was sheared using focused acoustic energy (Covaris LE220) to an average fragment length of 150 base pairs. Sheared genomic DNA was prepared for exome capture using a custom reagent kit from Kapa Biosystems using a fully automated approach developed in-house. A unique six base pair barcode was added to each DNA fragment during library preparation to facilitate multiplexed exome capture and sequencing. Equal amounts of samples were pooled and then exome captured on the xGen design available from IDT with some modifications. Multiplexed samples were sequenced on an Illumina v4 HiSeq 2500 using 75bp paired-end sequencing.

Raw sequence data generated on the Illumina Hiseq 2500 platform were uploaded to high-performance computing resources at DNAnexus (DNAnexus Inc., Mountain View, CA, USA), and an automated workflow was used to convert raw .bcl into annotated variants. It was handled as a call. Raw reads were assigned to the appropriate samples for analysis based on sample-specific barcodes using CASAVA software (Illumina Inc., San Diego, CA, USA).

Sample-specific reads were then aligned to standard sequences using BWA-mem (Li and Durbin, Bioinformatics, 2009, 25, 1754-1760). This generated a binary alignment file (BAM) for each sample with both the reads for that particular sample and the genomic coordinates to which each read was mapped. After alignment, the reads from the sample were evaluated to identify and flag duplicate reads using the Picard MarkDuplicates tool (picard.sourceforge.net), creating an alignment file with each duplicate read marked. was created (duplicatesMarked.BAM).

Subsequently, each Alignment of samples and local realignment of overlapping marked reads were performed. The GATK HaplotypeCaller is then used to process the realigned overlapping reads and identify all exon positions where the sample changed from the genomic standard, including single nucleotide variants and INDELs, and any positions where a particular sample differs from the standard. The zygosity of the variants in the sample was identified.

Counts of reads assigned to both standard and alternative alleles, genotype quality, which indicates confidence in the genotype call, and overall quality of variant calls at that position were output for every variant position. Variant Quality Score Recalibration (VQSR) from GATK was then used to assess the overall quality scores of variants in samples using the training dataset to access and recalculate these scores to increase specificity. Metric statistics were captured for each sample to evaluate capture performance, alignment performance, and variant calling. After completion of cohort sequencing, a project-level VCF is generated by joint-genotyping using GATK to genotype and genotype all samples at any site where any sample in the cohort carries a variant from the reference genome. and associated metric information was generated. It was a project-level VCF used for downstream statistical analysis. In addition to VQSR, variants are annotated with the Quality By Depth (QD) metric using GATK, with a QD greater than 2.0, a miss rate less than 1%, and a Hardy-Weinberg equilibrium p greater than 1.0×10 ^-6 . Biallelic variants with -values were retained for further analysis.

Prior to downstream sequence data analysis, samples with reported sex inconsistent with genetically determined sex, high rates of heterozygosity, low sequence coverage (defined as 20× coverage of less than 75% of target bases), or abnormally high levels of Samples with cryptic relatedness and genetically identified sample duplicates were excluded.

Sequence variants were annotated using an annotation pipeline using ANNOVAR (Wang et al., Nuc. Acids Res., 2010, 38, e164) and other custom algorithms for annotation and analysis. Variants were classified according to their potential functional effect and then filtered by observed frequency in publicly available population control databases and databases to filter out common polymorphisms and high frequency (presumably benign variants). An algorithm for bioinformatics prediction of the functional effect of variants with conservation scores based on multi-species alignments was incorporated as part of the variant annotation process and used to determine the potential deleteriousness of the identified candidate variants.

Example 3: B4GALT1 rs551564683 N352S frequency is abundant in the Amish

Through exome sequencing and linkage analysis in approximately 4700 Amish subjects, rs551564683 on chromosome 9 was found to be significantly associated with total cholesterol levels (p=1.3E-10) (see Figure 4). RS551564683 encodes a missense variant in which serine is changed to asparagine at position 352 in the B4GALT1 protein. The next most significantly LDL associated variant in this region was rs149557496 with a p-value of only 10 ^-5 , suggesting that the N352S variant is the causative variant. Referring specifically to Figure 4, in the exome sequence data, the variant at highest LD with Asn352Ser B4GALT1 was rs149557496 with P-value of HRCT1, 2.8Mb distance, R ² 0.78, P-value with LDL in Amish of 10 ^-5 . Whole genome sequence data in the Amish (TOPMED) failed to identify variants more highly associated with LDL-C in this region.

Further analysis showed that the B4GALT1 N352S variant frequency was >1,000-fold more abundant in the Amish population (see Figure 5). These data indicated that, in a cohort of 4725 Amish, 548 carriers were identified as heterozygous for the rs551564683-containing allele, and 13 carriers were homozygous for that allele (see Figure 5). As a comparison, a collective data set of another population cohort available to researchers - a total of 125,401 individuals - was analyzed, and only 79 heterozygotes and 5 homozygotes were identified in this collected data set. The allele frequency in the Amish cohort was predicted to be approximately 0.06, compared to approximately 0.0025 in the collective data set (see Figure 5). It is believed that genetic drift may explain the higher frequency of these alleles in the Amish.

Example 4: B4GALT1 N352S is associated with reduced serum lipids and increased AST.

The association of the B4GALT1 N352S variant with various phenotypes, including serum lipids, coronary artery disease (CAD), and liver traits, was evaluated. Linkages were performed based on an Amish cohort with individuals homozygous for the standard allele, individuals heterozygous for the alternative allele, and individuals homozygous for the alternative allele. Genotyping mean and risk of CAD for lipid and liver traits were determined, with effect measures adjusted by removing the effects of subject age, age squared, subject sex, and study (as phenotypic data were collected from several studies over several years). because). For pericardial fat, genotyping means were further adjusted for BMI. The effect size of variation on the measured phenotype was estimated at a 95% confidence interval. Characteristics and results are provided in Figures 6, 7 and 8.

As shown in Figure 6, the presence of the N352S mutation was generally correlated with reduced serum lipids, especially total cholesterol (p-value 1.3× ^10-10 ) and LDL (p-value 1.8× ^10-9 ) levels. , which achieved strong statistical significance. Individual heterozygosity and homozygosity for these alterations showed reductions in LDL levels of 17.3 mg/dL and 31.2 mg/dL, respectively. There was a trend between the variants and reduced coronary artery calcification. Additionally, the presence of this variant was correlated with increased aspartate aminotransferase (AST) levels (p-value 6.0×10 ^-8 ). The recessive model p-value for AST levels was determined to be 9×10 ^-23 . These mutations did not correlate with increased alanine aminotransferase (ALT) levels, alkaline phosphatase levels, or liver fat levels. Cholesterol, LDL and AST levels are graphically shown in Figure 7. In Figure 7, levels of cholesterol, LDL, and AST were measured in subjects homozygous for the standard allele (TT), heterozygous for the alternative allele (CT), and homozygous for the alternative allele (CC). ) refers to an object. The values shown are not adjusted. Values were recalculated based on subject age and adjustment for squared age, sex, and study (as tabulated at the bottom of Figure 7).

The effect of the N352S change on lipid subclasses was also assessed. These results are shown in Figure 8. Linkages were performed based on an Amish cohort with individuals homozygous for the standard allele, individuals heterozygous for the alternative allele, and individuals homozygous for the alternative allele. The results in Figure 8 show that the B4GALT1 N352S alteration is associated with a reduction in all lipid subclasses tested.

Example 5: With reduced fibrinogen levels B4GALT1 N352S association

The association of the B4GALT1 N352S variant with fibrinogen levels was also assessed in a subset of samples. For the serum lipids, CAD, and traits assessed in Example 4, the association with fibrinogen levels was found in individuals homozygous for the alternative allele, heterozygous for the standard allele, and homozygous for the alternative allele. The study was conducted based on an Amish cohort with zygotic individuals. Genotyping means for fibrinogen levels were determined in two subgroups of subjects - those not on clopidogrel therapy (drug-naive) and those on clopidogrel therapy (on-clopidogrel), and as part of the analysis, subject age, age squared. , the average level in each group was adjusted by removing the effects of subject gender and study. The effect size of variation on fibrinogen levels was estimated at 95% confidence intervals. As shown in Figure 9, the presence of the N352S mutation was associated with reduced fibrinogen levels in the drug-naive (p-value 1.15×10 ^-3 ) and clopidogrel-on-clopidogrel (p-value 2.74×10 ^-5 ) groups, respectively. . The drug-naive subgroup showed a fibrinogen reduction of approximately 24 mg/dl (see Figure 9). The on-clopidogrel subgroup showed a fibrinogen reduction of approximately 32.5 mg/dl (see Figure 9).

Example 6: Additional B4GALT1 N352S association

Within the Amish cohort, an assessment of the association between the B4GALT1 N352S variant and other traits including creatinine level, predicted glomerular filtration rate (eGFR), basophil level and hematocrit percentage was also performed. As shown in Figure 9, this variant was weakly associated with small increases in creatinine levels, but not significantly associated with eGFR, basophil level and hematocrit percentage.

Example 7: B4GALT1 ortholog knockdown in zebrafish

Similar to the cell-based assay, a zebrafish model was performed to investigate the effect of B4GALT1 p.Asn352Ser on LDL.

Zebrafish preparation, morpholino injection and validation.

Wild-type (Tubingen) zebrafish stock was used to generate embryos for morpholino injection. Adults were maintained and raised at 27-29°C and embryos were raised at 28.5°C. All animals were housed and maintained according to protocols approved by the University of Maryland Institutional Animal Care and Use Committee. Morpholino antisense oligonucleotides (MOs) were obtained based on previously published MOs targeting B4GALT1 (Gene Tools, Inc.) (Machingo et al., Dev. Biol., 2006, 297, 471-482). MOs were injected at 1 to 2 cell stages and verified by qRT-PCR quantification of wild-type B4GALT1 transcript. Off-target toxicity was measured by qRT-PCR quantification of the delta113 isoform of p53 (Robu et al., PLoS Genet., 2007, 3, e78). For mRNA rescue experiments, human B4GALT1 mRNA was transcribed from pCS2 ⁺ plasmid vector containing the open reading frame (ORF) of the wild type or N352S variant of the gene. mRNA was mixed with various concentrations of MO and co-injected into 1- to 2-cell stage embryos. For each injection experiment, a total of 200 to 400 embryos were injected, and each experiment was repeated at least three times.

LDL quantification in zebrafish.

One hundred 5 days post fertilization (dpf) laba were homogenized according to the experiment in 400 μl of ice-cold 10 μM butylated hydroxytoluene. The homogenate was filtered through a 0.45 μm Dura PVDF membrane filter (Millipore) prepared for lipid extraction. Homogenates were processed using HDL and LDL/VLDL cholesterol assay kits (Cell Biolabs, Inc.) according to the manufacturer's protocol. After precipitation and dilution, samples were analyzed by fluorescence analysis using a SpectraMax Gemini EM plate reader and SoftMax Pro microplate data acquisition and analysis software (Molecular Devices).

A genomic knockout of the zebrafish ortholog (B4GALT1) was generated using CRISPR/Cas9-mediated targeting of exon 2. Consistent with mouse reports of embryonic lethality in knockout animals, injected F0 animals did not survive to adulthood and consistently died in adolescence. To circumvent the lack of viability, a knockout approach using previously reported splice-blocking antisense morpholino oligonucleotides (MOs) injected into embryos (Machingo et al., Dev. Biol., 2006, 297, 471 -482) was used. The efficacy of MO was verified by qRT-PCR at two different concentrations (see Figure 10) and the possibility of off-target toxicity was excluded (see Figure 11). To quantify changes in LDL levels, 8 ng of MO was injected, the injected embryos were cultured until 5 (days post fertilization: dpf), and embryos were cultured at this stage according to a previously published protocol (O'Hare et al., J Total LDL was tested according to Lipid Res., 2014, 55, 2242-2253). Consistent with a role for B4GALT1 in LDL homeostasis, a significant reduction in LDL was observed in MO-injected Laba compared to control Laba (see Figure 12). These results were confirmed using a second splice-blocking MO targeting exon 2, which produced a decrease in LDL concentrations upon injection of 2 ng of MO (data not shown). To verify the specificity of these observations in zebrafish and to test the function of human B4GALT1, full-length capped mRNA encoding the human gene was subjected to in vitro transcription from the pCS2 ⁺ plasmid carrying the open reading frame (ORF) of the human gene. created by. To assess the ability of wild-type human mRNA to rescue the knockdown phenotype, it was co-injected into embryos with B4GALT1 MO and LDL was assessed in fasting laba. Three concentrations of mRNA (10 pg, 25 pg, and 50 pg) were co-injected with 8 ng of MO. Co-injection of 50 pg of B4GALT1 mRNA resulted in LDL levels that were statistically indistinguishable from those in laba injected with control MO alone (p-value = 0.14), suggesting that human mRNA could rescue the effect of knockdown of the zebrafish gene. (See Figure 12; Laba was treated with MO against B4GALT1, MO co-injected with WT human B4GALT1 mRNA (WT rescue), and MO co-injected with B4GALT1 mRNA encoding the Asn352Ser mutation (N352S rescue). ).

These data support the use of this system for functional interpretation of variants in human B4GALT1 and suggest that human wild-type B4GALT1 mRNA is functional in zebrafish with respect to regulation of systemic LDL levels. The impact of p.Asn352Ser on B4GALT1 function was further investigated. Site-directed mutagenesis (O'Hare et al., Hepatology, 2017, 65, 1526-1542), T to C changes were introduced into the coding sequence of the human B4GALT1 ORF construct to generate full-length mRNA. Co-injection of B4GALT1 p.352Ser mRNA and MO resulted in a reduced ability for rescue of the LDL phenotype. The resulting LDL concentration was 15% lower than that resulting from co-injection of wild-type mRNA and MO, a statistically significant effect (39.9 μM compared to 46.6 μM, p-value = 0.02). This level of LDL was also statistically higher than B4GALT1 MO alone (p-value = 0.01) (see Figure 12), suggesting a partial defect in function introduced by the missense variant.

Example 8: Targeted genotyping

Targeted SNP genotyping using the QuantStudio system (Thermo Fisher Scientific) was performed on 3,236 OOA subjects. Based on the LD structures of 14 SNPs, 7 SNPs were selected for genotyping, and the linkage evidence for rs551564683 was 4.1E-13, while it was about E-10 for other SNPs (Figure 14), which is This confirms that rs551564683 is a causal variant in this region.

Example 9: B4GALT1 N352S results in reduced enzyme activity in the absence of changes in protein stability or cellular localization

Investigation of the properties of B4GALT1 was performed in COS-7 and Huh7 cells overexpressing human epitope-tagged Flag-B4GALT1 352Asn or epitope-tagged Flag-B4GALT1 352Ser (Figures 15 and 16). Referring to Figure 15, confocal microscopy images of Flag-352Asn or Flag-352Ser using B4GALT1 or Flag antibodies show the same staining pattern (scale bar = 10㎛). Referring to Figure 16, intracellular localization by indirect immunofluorescence in Huh7 cells showed co-localization of endogenously expressed B4GALT1 and TGN56, Golgi apparatus markers. A similar co-localization pattern was observed whether human epitope-tagged Flag-B4GALT1 352Asn or epitope-tagged Flag-B4GALT1 352Ser was overexpressed (Figure 16). Referring to Figure 16, endogenous B4GALT1, Flag-352Asn, and Flag-352ser overexpressed in human hepatocellular carcinoma Huh7 cells co-localized with the trans-Golgi network marker TGN46. Confocal microscopy images of endogenous B4GALT1, Flag-352Asn, and Flag-352Se subcellular localization in relation to the trans-Golgi network marker TGN46 are shown (scale bar = 10 μm).

COS-7 cells were observed to have low content of endogenous B4GALT1 (Figure 17, panel B), and therefore this cell line could be used to detect missense mutations on protein stability and/or steady-state levels, and galactosyltransferase activity. The effect was evaluated. These results showed that the missense mutation did not affect protein stability and/or steady-state levels (by Western blot) (Figure 17). 17, the effect of 352Ser on protein stability and/or steady-state levels is shown. Panel A shows COS7 cells expressing 352Asn or 352Ser Flag tag protein fusions with free EGFP in COS7 cells. Cell lysates were analyzed by Western blot for B4GALT1, Bactin, and EGFP using commercial antibodies. Represents one of four similar experiments. Panel B shows mRNA expression levels for the B4GALT1 gene determined by RT-qPCR analysis. Data represent the mean ± S.E. of four experiments.

To determine the catalytic activity of 352Ser, untransfected COS-7 cells and COS-7 cells transfected with expression vector alone or expression vector containing the cDNA insert of wild-type or mutant B4GALT1 lysate were incubated with galactosyl. Transferase activity was analyzed. When normalized to the expression of FLAG-tagged proteins (Figure 18, immunoblotting experiments in panels A and panel B), the enzymatic activity of 352Ser was reduced by approximately 50% compared to 352Asn (Figure 18, panel C). . Referring to Figure 18, the effect of the 352Ser mutation on activity is shown. Panels A and B show COS7 cells expressing 352Asn or 352Ser Flag tag protein fusions in COS7 cells. Cell lysates were incubated with rabbit anti-Flag IgG or rabbit pre-immune control IgG. Immunoprecipitates were analyzed by Western blot for B4GALT1 or Flag using commercial antibodies. Represents one of four similar experiments. Panel C shows B4GALT1 activity in immunoprecipitates measured with a commercial kit (R&D). Each data point represents the average of the calculated ratio of B4GALT1 specific activity and the amount of 352Asn or 352Ser protein recovered in the immunoprecipitate. Signals from Western blot ECL were quantified densitometrically using ImageJ software. Data represent the mean ± S.E. of four experiments (*, p < 0.05, 352Asn vs. 352Ser).

These experiments show that this missense mutation has no effect on the level of protein expression and its localization, but it leads to lower enzyme activity.

Example 10: Carbohydrate Deficiency Transferrin Test for Congenital Disorders of Glycosylation (CDG)

CDG testing was performed using 0.1 ml serum samples from 24 subjects (8 minor homozygotes, 8 heterozygotes and 8 major homozygotes) from 3 genotype groups. Each minor homozygote was matched with a heterozygote and a major homozygote (who are siblings or closely related individuals of the same sex based on kinship coefficient). Age and carrier status were also matched for major lipid-modifying gene alleles in APOB ^R3527Q .

Water diluted samples were double washed using an immunoaffinity column. Glycosylation profiling of the eluted proteins was performed using a mass spectrometer operating at a 2 scan range specific for APOCIII and transferrin. Glycosylation deficiency was determined using the glycoform ratio of each protein. CDG testing was performed at the Mayo Medical Laboratory at Mayo Clinic.

Results showed that all 24 samples had normal levels of mono-oligosaccharide/di-oligosaccharide transferrin ratio, a-oligosaccharide/di-oligosaccharide transferrin ratio, ApoCIII-1/ApoCIII-2 ratio, and ApoCIII. It was indicated that it had a ratio of -0/ApoCIII-2. However, while all wild-type samples had normal levels of the tri-sialo/di-oligosaccharide transferrin ratio, levels in all heterozygotes were in the intermediate range, and levels in all heterozygotes were abnormal, with matched wild-type and was significantly higher than that of heterozygotes (p=7.6 E-10) (Figure 19). These results indicate that this missense mutation is associated with defective glycosylation as a result of reduced enzymatic activity of B4GALT1.

Example 11: Global N-linked glycan analysis of plasma glycoproteins

To determine whether desialylation and hypogalactsylation only affect transferrin or extend to other glycoproteins, global N-glycan analysis was performed by the analytical chemistry group at Regneron. carried out. Lectin-rich glycoproteins were extracted in replicates from five pairs of sera from major and minor homozygotes, and global N-linked glycan separation was performed on labeled glycans using hydrophilic interaction chromatography; Detected by fluorescence and analyzed by mass spectrometry (HILIC-FLR-MS) (Figure 20 and Table 5). Referring to Figure 20, representative HILIC-FLR-MS spectra of N-glycan analysis of glycoproteins from matched pairs of minor (SS) and major (NN) homozygotes of B4GALT1 N352S are shown. These results show that heterozygotes have significantly higher levels of hypogalactosylated and lower levels of sialylated glycans (biantennary glycans with only one galactose and one sialic acid). (p=3.1 E-5), unsialylated biantennary glycans with only one galactose (p=0.001), and truncated biantennary glycans missing both galactose and sialic acid (p= 0.005)). On the other hand, heterozygotes had significantly lower levels (p=0.001) of biantennary glycans with two galactoses and two sialic acids (Table 5). There was significantly lower galactosylation (p=9.2 E-5) and sialylation (p=0.001) among heterozygotes, while there was no difference in fucosylation levels (p=0.5). Both CDT and global N-glycan analysis of serum show significantly increased levels of carbohydrate-deficient glycoproteins in heterozygotes, indicating that B4GALT1N352S leads to defective protein glycosylation.

The present disclosure is not limited to the embodiments described and illustrated above, but is subject to variations and modifications within the scope of the appended claims. The disclosure is also not limited in any way by the use of any headings mentioned herein.

SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. University of Maryland, Baltimore <120> B4GALT1 Variants And Uses Thereof <130> WO/2018/226560 <140> PCT/US2018/035806 <141> 2018-06-04 <150> US 62/515,140 <151> 2017-06-05 <150> US 62/550,161 <151> 2017-08-25 <150> US 62/659,344 <151> 2018-04-18 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 56718 <212> DNA <213> Homo sapiens <220> <223> wild-type B4GALT1 genomic sequence <400> 1 gcgcctcggg cggcttctcg ccgctcccag gtctggctgg ctggagggagt 50 ctcagctctc agccgctcgc ccgccccccgc tccgggccct cccctagtcg 100 ccgctgtggg gcagcgcctg gcgggcggcc cgcgggcggg tcgcctcccc 150 tcctgtagcc cacacccttc ttaaagcggc ggcgggaaga tgaggcttcg 200 ggagccgctc ctgagcggca gcgccgcgat gccaggcgcg tccctacagc 250 gggcctgccg cctgctcgtg gccgtctgcg ctctgcacct tggcgtcacc 300 ctcgtttact acctggctgg ccgcgacctg agccgcctgc cccaactggt 350 cggagtctcc acaccgctgc agggcggctc gaacagtgcc gccgccatcg 400 ggcagtcctc cggggagctc cggaccggag gggcccggcc gccgcctcct 450 ctaggcgcct cctcccagcc gcgcccgggt ggcgactcca gcccagtcgt 500 ggattctggc cctggccccg ctagcaactt gacctcggtc ccagtgcccc 550 acaccaccgc actgtcgctg cccgcctgcc ctgaggagtc cccgctgctt 600 ggtaaggact cgggtcggcg ccagtcggag gattgggacc cccccggatt 650 tccccgacag ggtcccccag acattccctc aggctggctc ttctacgaca 700 gccagcctcc ctcttctgga tcagagtttt aaatcccaga cagaggcttg 750 ggactggatg ggagagaagg tttgcgaggt gggtccctgg ggagtcctgt 800 tggaggcgtg gggccgggac cgcacaggga agtcccgagg cccctctagc 850 cccagaacca gagaaggcct tggagacttc cctgctgtgg cccgaggctc 900 aggaagtttt ggagtttggg tctgcttagg gcttcgagca gccttgcact 950 gagaactctg gtagggacct cgagtaatcc actccctttt ggggactgac 1000 gtgaggctcc cggtggggaa ggagactgac ctctcggttc acgtgtcttg 1050 ccatagagcc actctcctga gtgggttttt ctcctgatcg tttgggccaa 1100 gtgacttctc tctgaacctc atatttctct tctggggataa taaatggtca 1150 ccctttcaag gggttgtttt ggaagatatt gtgaacaatg gtaaataagg 1200 gcttaattaa tgagggtaag ccctcagtaa attgtcactg tgtgttcatt 1250 tcttcctctg tgtggatcgt gaccgagagc ccttccccct agcctcctcc 1300 tggtatgggt acccaaaacc taggtgagca gggatctctc ccaggggcag 1350 agagcttgtg tactctgggt gttagagggc taaaatataa ccagtcaaca 1400 ccacgttgcc catttctggt acttccggta gcagcctgag tctcaattat 1450 cttgcccaga tgatctgaac tctgacctct agcctgtttc agcataggca 1500 gagagcttga gtaggtgagt ttgcattcct catagcagct ggctgagcct 1550 agtctggact tctctttgac ctgtaaccta caggcccaca ggcccaaggc 1600 aaccacaggt tgcttccagg gttaccacac aggtggtttc tcatttctaa 1650 tgctaggttt tagataattg ttgtaagtga ggggccctgg caggcaggat 1700 gacatcctgc caataggagt tttctgtcac tttcccacag agccctggct 1750 actacatact cttgctcaat ttcgccagta attgcgtcaa tgtgttcata 1800 tcaagtttgg gaagaacatc ttggaattgg tcagacgtga actgtggtaa 1850 taatgggggc ttgttttttt aagcagataa ttaaattcct ttgcatttga 1900 tgattattct gggaagcaga ctagtcccat aaaatgaaat ggactctgcc 1950 ttgctgctaa gtgtctgact tgagacatgc tatcgagttt ctcaaaatct 2000 cttccttgtg taaaatgtgg ttgtcgatga ttaccttaca ggggtttttt 2050 taagactaaa tgagatcgtg tacattaaat acaggcactc aggctgggca 2100 tggtggctca cgcctgtaat cctagcactt tgggaggctg aggggagtgg 2150 atcacttgag gttaggagtt tgagaccagc ctggccaata tggtgaaaca 2200 ccatcccatc tctacaaaaa tacaaaaaag ttagccaggg gtggtggcat 2250 cgcagctact caggaggccg aggcaggaga attgcttgaa cctgggaggc 2300 agaggttgca gtgagtcaag attgtgccag tacactccag cctgggcgac 2350 gaagcaagac tgtctaaaaa aaaaaaaaaaa aaaaaaaata cgggcactca 2400 atacacgta taataataat atagtaataa tatttgctta ggatctttaa 2450 aaagtttcat tttttcagac tcccacagaa atggctctgc acagcagagt 2500 gaagggggag agagactgag tctccaggcc agaaaaaggc caggtttttt 2550 gcttttgttt ttagttgttg cctggatatt gcacagaaag aaaaaataat 2600 tagcaagtta aaaaaaagta ccgcaaagtt gattacattg gtatttgagt 2650 atcacatctt ctctcagaag cgtaagagac aaggtcgtga ccatacctct 2700 gcttagtttt gttttgtaat ggtgttgcta gtgatcggct tgtcaccagt 2750 tactggtgtt tctaaatgga ctataattgg ctacttgaaa ggacttcctg 2800 agaaagaaca ttttggagga cgaggagaga gtgccttctc tattttggct 2850 gctttcatgt gacatgcaag agaccatgac gtttaggctg ctgctgaggc 2900 agccccagaa atgggggccg agaggtcttt tcttcatttt aatagggtct 2950 gtaggtttgg gtggttaggt acagttctca gaatggaggt tcctggctat 3000 gaggccttga gaaagctgaa agtctccttg ggagtgtgtg ggtgggggga 3050 gtcgagccca tctgttcatg ggcaggtgtc agccaaagcc cttgcgggtg 3100 gttttgaggt tggtggggaga aagcatccgt ggggtttaga gttgtggcct 3150 tttcactact tgcagttcct ttccccgact tggctttact ttctggtgtc 3200 caggggtctg ggccagatgc tgagattcct ctcagctgac aggtgtgggt 3250 tatgggcaaa cccttccctg gaggacataa ggcaccggat tggactgctg 3300 atgggttgct gttggagttg tcagggcctt ggaaatagtct tcagatagac 3350 ttgggttagt gtgacctggg gcaggctgca ggtttggagc catagtaccc 3400 cccgccccca caccgggcac cctgctctgg gctaatgtga ggcttgcagg 3450 agtgagtgat gcagtgggaa ggggggcctt tcctgaggat tctacagctt 3500 tctccaggga atcctcccag gtagtttagg cctgcaggtg ctatgctatc 3550 cttctttcct aaccctgtct caggtcctca gcggggccat gcggcatcca 3600 cttataaccc tgcagcgagg ccctcttttc tggccacctg ggtgtttgcc 3650 tgctgagatg ggaggaacag tggccttggg cttcttcccc cgtcatgttt 3700 atctctgctc agatgggca gcagctcaat gggacttgac cagctgtggc 3750 actgccagtc tgaagatgag tagggtgatg gggggaggtg ggcagtacct 3800 gaagctgaac tggtgagaga ggcaggctgg cctgggggct cagctggggc 3850 ctgggatggt tggtacagtc ccctcagggg ggtaggggag tgagtgttag 3900 actgcttaag cctcagaggc cgctcttgcc cacctatgct ttgaggagat 3950 cctcttcatt tgttcaaagg gaagactctg atctagagat gggcacttgg 4000 accagcaaac agcagctaca ggtagccagg gcacccgagg agcacttgct 4050 catgagccgg tttccctggt ttttatgggg gctgttgctg agcgtctgcc 4100 agggtttgtg tcctagcact tgctggtctt tgctgggctc tcagctctca 4150 ggtgtttctc taccagcacg tttccccctc cctcatatgc acacatgtgg 4200 acacaagcag gctgcccagg acagagtgta ctttgaggct tgggaaagga 4250 ctctctctcg cccttttggg gatgagcctt ggaacctcat caccttccgg 4300 cttggggtgg agcttcatcc tgggggttga agctttaggc tcagataact 4350 agtcttgtaa gccagttttg tcctgttgtt tttttcgtgg aaaataatgt 4400 attgacgtat acacagacat tctttgtcta acagtctgag attgagaaat 4450 accctccatg actatttggt ttgctttcat ggtgaaactt ggtcgctttc 4500 ttagacacag cctatggcaa taagagtgat ccctggctgc tgtaattcat 4550 tccagacttt gagcaaacac aaggcaccgc ctccacctgc agtggagcct 4600 ctgatgaacc aaatggaaac tccttgggga atggggagta agagccaaat 4650 gtgggattgg acttaaactg cagcttctta gaactgtagc attccacgat 4700 gggattgtct agtgctcttc ctggaggtta ctattcaata gttggctagt 4750 gcacaggttc aggggtgacc tgatatgccc tagcgtttca gaagatccct 4800 gcaaggtgtg tcttttggtc catctgaagg gtcttgtatg gtgatcttgt 4850 atggatatcc gtgacggcta aggcatctga taacttcatt ccttcagttc 4900 cagcagtgtt cctgtattat gctgggcact agagctacaa agaagaaaac 4950 aaagtgcctc ctcttcagga actcttaatt taggcagggg aggcataatt 5000 gaacagtgct gaggtcatct aggggaacca aagtgtgtat ttatcccctt 5050 ccctatcact cccctccctc cttcatttct tcctttcttc tttcagaaac 5100 tccaagttca tatcaaaatt ctccagccct ggttttatattt ggttgtgtga 5150 aaattttcct ctaatttctg aagctatgca ttagttctgc tgagtaatct 5200 ttaacttgct gctttataat gattataatg agatatcact gggtattatg 5250 gtctttgggt agcagcaggg tagggatttc caggctggga ctaagctaat 5300 ttatgggttg ggaattatgg ggcagttaat agcaaggcag tccaagcttt 5350 ccacagattc caccctaggg accatccaga cttaaggaac agggccggca 5400 ggctcatccc ctttgcactc agctgggcta tgggtgtgtg tttgtgaaag 5450 aggtttattc agtagtcata cctgctgatt tccctgctat ctgtttaccc 5500 agtgcctcct gtaccttgtt tcttactctt tgttctctgc tcttactatg 5550 aagaagcaga gactggaatt ctgcttgaac ccacatctac ctggaaattc 5600 cagtttttct tgtccagtgg agcagcaatc cagttgtttt aggacaaatg 5650 gtctgccctt gaagcttaaa tcctttgagg gcctggcatg gtgacagttt 5700 tacatttggc tttggtatag actggtgtgg tccctgggca gtgaggtcac 5750 tgtaaggcca gccagccaga ccctggctcc taggggaatt aacaaggcat 5800 gggattagac tcacagggtc cctcctgtcc ctaaacttgg taggggttcc 5850 tgggagccag actgcgatta agattgtaga gacctgagac ctgagttgta 5900 ggggcctctg tgttgatctg ggccattgcc gggtgagctg aggcggtcac 5950 tagctcaagg agtgatctca ggatattgtt ctgtaagtca gagacctcca 6000 ggttggagag tggggcttgg gggtggggga cagggtttag tggggagctg 6050 gttctgggtg aatgtggcct aaagggattt gtccttagaa gacagagggg 6100 tgagtcacac actcagtgct tcaggttcca ctttgcggct tggcctcagc 6150 ccgccccttc cctgcacaaa tgaaggccag gggctatata attggctgtt 6200 gctgaattct ttggcagtga ttttaaagtc tggtctgggt gtgttatgta 6250 gctgcttctc tatccactcc ccacacccgc tgcttctcca gagcccctca 6300 caaagcccag gcagagagag agagagagag agagagaatg acttgcctca 6350 cagagatgtt ggggatagg ataggggtat gggtctttgc ttttgccttt 6400 tgagggggga taatctcttc cttcatttta aaagtaaaaa gtaatgcagg 6450 ctcattgaaa ataatttgaa aagttgaaag agatataaaa gcacacccaa 6500 attcctatca cccaaaagaa acataccggc atatttccta ctagtctttt 6550 tcatgtttaa gaatatagct gatatatttt tttttctttt tctttttgag 6600 acagggtttt tgctctgtca cccaggctgg agtgcagtga tcacggctca 6650 ctgcagcctc gacctctcgg gctaagcgat tctcccactt cagtctcccg 6700 agttgctggg accacaggtg cacaccgcca tgcctgacta atttttgtat 6750 tttttgtaga gatggggttt tgccatgttg cctaggctgg tctcgaactc 6800 cagagctcaa gtgattcacc tgccttggcc tcccaaagcg ctgggattat 6850 aggtgtcagt caccacaccc agtgttatag ctgttgtctt tatagatgaa 6900 cagatagatt gacatagatt catgtagata gcctggtgtt cagcattttt 6950 catttaagat tctgtcacag acttgaccct atacctttaa aaatcacaaa 7000 ggcagtatca tagtctgtca gctgaatatg ccataactta aaaaaatcat 7050 tcaactgttg ctgaacacac acatatacat atatagtttt tgttttttct 7100 tagtgatgta gtgatgcttg tgcagaaagc tttatgtact ttttggatgg 7150 tttctgtagg agagctttct aaaaaagggaa aaaaagtgtt gaatgttttt 7200 tgagaagggc tagattttca agccagtctt acaaaagggat agactcattg 7250 gaaattccag atttgcttag tgctggcaga tgagtatcac ttattgctga 7300 acaatgtgtc tagaattctg attaaaaaag aaactaggtc caggaagtgc 7350 ctgggggcag gggcaaaggg ccaggctgca ggataggctc ttaggatctg 7400 gctgagcaga aatctgctgt gaacagaatc ggtgggggtg atgctttctc 7450 agtaacttct ccatttgttt ctttagcagc taagtccctg tgctggactt 7500 ctgtggacta ctgtggctct ggggctgtgg ttgtgggtga acaacagcta 7550 gctaaaccag tgctgttgac atcattgaga tgtgacgcac aggaaggtgg 7600 gagcaagctt gcaaatcaga ttctgaaaca tatagcacag ctctcccacc 7650 tccaggtggt cctgagatct agggaggagc catagtgaga aactttaggt 7700 ttctaggaat tctcttaggg agaagctctc ttagggagag gcagaacctg 7750 gttctcagtt ggggctgatt caggtgggtt agatcaataa agcctcaggc 7800 cagtgtgcca ggctattccc aaggagtata ctttgaagtt actcccttta 7850 gaatgtcctc agtggagata aattctctct gaggagcagt tttgtctgcc 7900 ggggtcattt ggcacaaagc ctggagtgct agggcgaggt tgcactgagg 7950 gaagggggcag gattatgtca gcagtgtgac ggatacagtg tgaggtcagg 8000 ctccttcctg ccccaccacg ggggcctaga ggtcatgggg agggtccctg 8050 gcaggggatt caatcattgc ttggccccat gacagagtat attctaaaaa 8100 tgccttaagt ttttttcttt caaagtttct tcctgttttg cataatggcc 8150 ttttgccttt gacatcctga aaccgcagag ctgtcattgg tgttgcagga 8200 cactgccagc ttgaaaaaaa tcaacaaacaa aaaaagaaac aggaaaggat 8250 gtggagttca gggtgcggcc tagggaagct ggtatttgcg ttatgggatt 8300 gtggggatgt ggtattaagg tgttgggtag cgcctgacat ttagaggagt 8350 actctgggca gagtccctgc ctgcccaaga ataggtagaa ttgagtcttc 8400 acaccaaagt caggagagac cccctccccc caggaagaga atgaacaggg 8450 actcatttcc tcattcagca aacttttat ggtaactaca ctatatgaag 8500 tgtgagagat agacatgaac aagagaggcc cccactcttg ggcagtccct 8550 tagtagtagt agatagactc tggcaatatg gtgtggtcag agagaggaag 8600 cctgggtgct ttgagggtac tgaggaggtg cagggagcca aatgggtggt 8650 ctgggccagg gccagagtca gaatgaagga cctctcttcc agacgttgat 8700 tttagcatct ctgtctctca gtatgtttga acagtctccc ttattggaag 8750 ggcaggagtc tactgctaaa agtaacctgc gatttcctct acttgctgtc 8800 atgtggaaag aatactaaag ctgaaattcc aaaagttgca cacctttacc 8850 agcagggcag gagaggaaag gaaatggagg cagagtgagc tgaagatgat 8900 aaaagaaaga gaaggtggtg cagtttggac tgttatggac agaggaagtc 8950 tgagggtagc tggactgagg gatcaaaggg aggcagttga aagggaagag 9000 agctgcagag agggatttct tggtctgcag agggtaggag caagccttga 9050 aggctgctgg agtgaggatt ccgagccctg gtctttattc tttttctaat 9100 tcatttacatc attttaggca agtcctaact cctttggtct ctgttgtctt 9150 tctgaaattt gagtgggctg ggcctgctgg tctttagcct ctgtctttct 9200 ctacctccta gattccagtt tggcgagtgg gggggaaaac ctggttgtat 9250 atgcaacgtg aaaggcctct ggaattcctt ttgaagctca ctacccatga 9300 ggcttctgct aaggatttca tcatgtctgt ctaagcagac ataaaaattt 9350 tagcaggtgg atgacccgta gaaatggcac aaggaatgtt tctttctgtc 9400 acactgtggt atttgattta agaaagttgt tatcctctct gtgcctcagt 9450 gttctcactt gtaaaatggc aataacagta tccacctcat agatgttatg 9500 aaatacaggt agtagccacg aaagggctta aaacagtgcc taacacagaa 9550 taagttgtga atatatgtta tttattatg gtagtataat gcttatttgt 9600 gaagattttg gcttttgctt tataggacct tttttttttt tagttgaaaa 9650 tacaatgtta ccatgttaaa tgttaaaaaaa aattctactt accattgtaa 9700 cagaacatgc tcccacttct gtaacagagc ttgctattac ttttcaaatg 9750 catacatatt ccaatgcata tattccaatg cagttgtaga gtgaaactgt 9800 ttgcatgcag ccatttttat ccaaacattat cttataaaat gttatgttgt 9850 ttatgattat cctaattatc ttttgttgct gtctagtatc cttatagata 9900 ttccattagc atacactatt ccaggtttca ctatcgtcga taatctagat 9950 atgaacattt ttgtagtgtg tagctctttg cttcagttga attactttcc 10000 tgggataaat tcctggggaa gaatttctag gccagaggat atggtcatct 10050 tgacaatact gattcacatt gctgcattgc tttccaagag gtttggaatc 10100 attcacaggt tctaaattgg aaaatcctgg cttttgaagt atgtggattc 10150 taagggcgat ttggatctag ctggagcctc acactgacac ttccagccag 10200 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtagt tccctatgct 10250 ggacaccgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtagttc 10300 cctatgctgg acaccatgtg gcctttctgg acattagggt tttcctgtga 10350 ttgcctcaga gcagttcctg ttgaattcac tctgtgtcca caaaaggagc 10400 cttactgtgg ctctttcaac acccacctac ctttgccaag ttggtttaca 10450 gaaagtaaga acattctttc cttcttcctt gatatgtggc gctaaaaccta 10500 tagcatgggg caggctctgg ctttaaaaac ctgacttaaa aataatggtg 10550 ttgatcaaaa agtttgtgga tcagtttttg gaaacactgc atgtagccat 10600 ccatagaaac ttatattctg ttgggctagc ctgggcgcct gatcatttaa 10650 ctcatgtgga tgaacttcta tgtaatagcc ctggtgtatg ggatccagaa 10700 acagggccct aatgaagaaa ggcttttaaa ttatgttgga taaaaataag 10750 ttgttacaat agcccaaagt ctgcaaatat gaattgccag ttctgtcctt 10800 gtagtcatcc accatgtgcc tgcatctttt gtagactctt gtagattcag 10850 aagcccactg aattgcataa atgatggaat gattttagac ttagtgattt 10900 cagtgactaa aagtttacag atcctggccg ggcacagtgg ctcacaccccg 10950 tattcccagc actttgggag gccgaggtgg gtggatcacc tgaggtcagg 11000 agtttgagac cagcctggcc aacatggtga aaccttgtct ctactaaaaa 11050 tacaaaaatt agccgggtgt ggtggcatgc acctgttgtc ccagctactt 11100 gggaggctga ggtggggagaa tggcttgaac ctgggaggcg gaggttgcag 11150 tgagcccaca tcaggccact gcactccagc ctgggtgaca gagtgagact 11200 ctgtctccac ctcccccgcc ccccgaaaaa aaaaaaagtt tacagatcca 11250 gcagatgggg catattcaat ttgtgacagc cactcccttc accttatagc 11300 tatgtcatat gtcttcttct cctttgactg cattctgcag cagtcagttg 11350 tgacttaata tggcactctg ggcccactga attaggtcag agctgctagt 11400 agtatattgt tcctagagac ctagggcaag attttcttac tacataaaat 11450 gagggagata atttcttacc tcaagatgtt ggtaagagga gtgaatgagg 11500 ttagttatat ggtaatatca gtactctgaa tgtcttttga tcaatgccta 11550 actcatcttc ttgggcacaa aaggcataca gtcagcaccc ttaggccaca 11600 tataaaattc ctccaaatgc aggttttcat ctgccttggg gcagagtcaa 11650 gagaaagaag aggaagaggc gtgaggctct gaccacaact tagggacaga 11700 atatagccca aagcgagtac cccaggccac aaggagaagg ccgctatctt 11750 gttgaatcca cagcactgga aacttggagt gtgtgttccc ctgtgtcagt 11800 tacactggaa ttttatggct gctcacattc ttcccttcag gtggacgttg 11850 ttcatcagta tcctgggcaa gaggccatca taaaccacag acagctgagt 11900 gattaggaag aggagctgaa gagggagcat tagatgtttg attgagtctt 11950 aggtgagaaa gtatatcatt aaaacaaaaa gatagatgta ggcgggctca 12000 gtcttgtgtg cctggtgtgt tggtagaaaa actaaagcac aagcctgtag 12050 ataacctgct ttattctacc tcggggctgg tgttggaatc caggatgcca 12100 gaccctaaag tccagctctc tttccaaacct actgaataat ccgagagaaa 12150 tcatgttctc tctctgggcc tcagtttgcc catgtataaa atgagatgaa 12200 ggattggctg ggatgctctc cagagtctct tcctgcctgg agttctgacg 12250 tagccatgta ctcctgctca gcatcgctaa atggctttgt ggtaggacca 12300 ttgagtgctg cctccattag ggccagctat gtaatgctgg ggtggctgtc 12350 actgggccct aagagccagg attggtctta ctggagaaat ccacatccac 12400 ctaaacttaa gacccagggg tgtccaatct tttggcttcc ccaggccaca 12450 ctggaagaag aattgtcttg gaccgcatat aaaatacact aattatagcc 12500 gatgaggtta aaaaaaaaaaa actcaatatt ttaagagagt tcatgaattt 12550 gtgttgagct gcattcaaag ccatcctggc cgcatgtggc ccatgggcca 12600 tcggttggac atgcttgctt tagacctccc agcaattcta gtctctaaac 12650 aggaaatcaa aagtcaagat gaatagataa gttggtcagt gtgaaaaagt 12700 aattggtggg agccactgta gatgcagggt tctaggctcc atcaacaacc 12750 acctacatca ctgaacgaaa gataatgctt gttcagcact tattacatgc 12800 caaccatggt aaaaatactt cagatgcatt gttttcatga actctcacag 12850 cagctctttt tcttgcctaa atgccccgtt agaacctcca gtacaatgtt 12900 aaatagatat gctaagagac aacatatgtg tcttgttagg gggaaaatat 12950 ccagtctttg actattaaga atggtgttag cagtgggttt ttcctaggtg 13000 ccctttatca ggttgaggaa gttcctttct attcctggtt tgttgagtat 13050 ttttatcatg aaaaggtgat gggttttgtc aaatgctttt ctgtgtctgt 13100 tgagatgatc atgttttttt gtcatttatt ctattgatat ggtatattat 13150 acattgattt ttcagatatt aatcttgcat acctgggata aatcccactt 13200 ggtcatggtg tataattctt tttattgtt gctggattga gtttgctagt 13250 attttgttga tttgtattca taacagatag tggtctgtag tctttccctc 13300 cctccctccc tccctccctc cctcccttcc ttccttcctc tctctctctc 13350 tctctcccct cccctccctt cttttcccct cctctcccct ccccttccct 13400 ttcttctctt tcatagttgt ttaccactgt cagaaaaggt ctgttcgttt 13450 tctttcgtcg tgagatcttt gtttggtttt ggtatcaggg taatactgcc 13500 tcaaaaaatg agtaggggaag tgttccttcc tcttctgtat tttgagagag 13550 tttgtggtcg gtttttatta attcttcttt aaatatctgg tagcgttcac 13600 cagtaaagcc atctgggcct gatgttttct ttgtggaaaa ctttttgatt 13650 cctaattcag tttctggtta taggtctatt cagaccttct attttttctt 13700 aagtcagttt tgatagtttg tgtcttccaa ggagtttgct tcatctaagt 13750 catctaattt gttggcatac atttcatagt gattccttat gatccttttt 13800 atttccgtta aagttggtgt agggatagtc cctctttcat tactgattat 13850 aataatttga attttctttt tttcttagtc ttgccaaaag cttgtcattt 13900 ttattgatct tttcagagga ccaactttga gttcattatt tgttctcttt 13950 gttcttattt ttctgcttca ttaacttctc taatctttat tctttcattc 14000 tgcttgcttt tggttaagtt tgctttttct ggtgtcttaa ggtagaaggt 14050 taggttactg atttgagatt taaagatcat gctctttaaa cgttttgata 14100 gatactgtca gtttgccctc tggctttttc tcattaacag tgtataggag 14150 tgcttattcc tcacactcat accagccctg ggtgttacta acctttatat 14200 atttgccagt atcatattca gacatagtat cttgttttaa tatgtttctc 14250 tgattactga tgaagttaag caaattttca cgtgtttat ggccatctgt 14300 ctttcttttt tcatcctttc tttcaagatg ggagtctttg ccatgttgcc 14350 caggctggac tcgaactcct gggctcaaat gatcttcctg cctcagcctc 14400 ctgagtagct gggactatag gcgtgagcca ccatggctgg cttgcccatt 14450 tgtatttctt atgtgagtat tttttctttt tttttgaagt ggagtctcac 14500 tccatccccc agagtggagt gcagttgtcc gatcttggct cactgcaacc 14550 accgcctccc aggttcaagt gattctcaca ccttagcctc ccaagtatct 14600 gggactatag gtgtgtgcca ccacacctgg ctaatatttg tatttttagc 14650 agagatgggg tttcaccatg ttggccaggc tggtttcaaa ctggcctcaa 14700 gtgattcacc tgcctcggcc tcccaaagtg ctggggattac aggtgtgagc 14750 cactgtgccc agctgacttt ttttttcttt tttttaaccc ttttttttt 14800 ttaccctttt tttggcccat ttttttttac cctttttctt ttaacccatt 14850 tttctattag ttttaaaaat atgtttgcag gagcttttta tattgtggat 14900 ttttcttgtt tattacatat catttgtaaa tatggtctct ccatctgtca 14950 ctcttcttta tctctggttt ctttagctat gtagaagttg ttatgttatg 15000 ttatgttatg ttatgttatg ttatgttatg ttatgttatg ttatgttatt 15050 ttttggagag ggagtcttgc tctgtcgccc aggctggagt gcagtggtga 15100 aatctcggct cactgcaacc tctgcctcct gggttcaagc gattctcctg 15150 cctcagcttc ccgagaagct gtgattacag gcacccgcca ccacacccag 15200 ctaatttttg tgttttagta gagacggggt ttcactatgt aggtcaagct 15250 gatctcaaac tcctgatctc aaatgatcct cccaaagtgc tggggttaca 15300 ggcgtgagcc actgcactcg gccagaagtt ttgaattttt atgtgtttaa 15350 atctatgttt tcctttatga cttcaggttg ctttcatact taagcaggtc 15400 ttcaccatcc caaaatgata aaatttttct cctgagtttt cttctaagtt 15450 ggttctttag aagccaccaa cttggcttcg acagcaaaag atgaacagaa 15500 tttctgttca actctcatgc tgcaagaagc tttatgtaat actccaggga 15550 ccctttaagg tcccagagtt ttcctccaaa tctatcagtg attctagtgg 15600 ctaagagtag aaatgtgaaa atttagccat gtgtgctgat agagctgtag 15650 taatttgtaa gctctgaagt tctaaggagt caggggagaa gggaaagtaa 15700 catttatga acatctatta gctcaataag aacatgcgat aagtatgtat 15750 atgtattatt tcacttacat ctgaaaggaa ggcataatta tccccactcc 15800 ttagagaagg aaattggagc tggctacatt taaagtagtc ctgacaccag 15850 agagatattg ccaggagtac ttggctggct gagtgcccag atggcccata 15900 ggagtagtgg gccctccaca gtccaaggtc tggttctagg tggagagaga 15950 aggatgtgct cgtagtcagc accgcagctc cagaaaatct gctggggctc 16000 caaaactgat tagaggggca gctgactcag taataaaact cccagggagac 16050 ttacttacat actggaatgc aaagttgcag ctttactggg aagattagaa 16100 ctgttatga gtagcttaga aatctctggc tgaattcact gcaaggggaag 16150 ccgcaggata agctaactgc tggtgagtca gcagtcagag cagggaagtg 16200 aatttaacat tagatgggtc agtctctcgt ggctgatgaa ttcatcccca 16250 caatactgta cacctgcctt agggaccttt gtctggacta ggggttgggg 16300 tccccctcct ttgtacagcc ctggaaggac acatccagct ccatccgcca 16350 tctctccctt acttatttcc ttccttcctt ccttctttcc atccagccat 16400 caagcttcct ttcatggcca ataatcatca ttggggtcta ctcatggact 16450 ctcttgcctc atgtatttgt tttattttgt cctcattccc acttctattt 16500 cccaggtata tcacaggcaa ctattctaac gtatttatag tttgtgtatc 16550 tgtttttgct cttgccaaaa tggaagccac tgctttatac atagatgtat 16600 tcttaacttt aaaaaaaatt tttttagatt aacctacaat aaaattggct 16650 ttttggcata tagtctataa attttaacac atacatattt ttgtgtatct 16700 accaccacaa tcaggataca gaacagttcc atcaccccaa aaaaatccct 16750 cttgtagtca cattctcctc ccacccttaa tcccaggcaa ccactgatct 16800 attcttcatt actattgttt tgtctttttg aggatgtcac ataaatggag 16850 tcacacagta tatatacatt tttttaaaca tatgtaaaatg gcattttata 16900 gctcattttg attatatgtt tttcatccag ttctgttttt tttttttatt 16950 tttaaaaagt ttgacataac ttcagactta cagaaaagtt gttagactaa 17000 tacaaagaat tcctggatat cctttggagt ccctaaatgt taacatttta 17050 ctatatttac tttttccttc tctctctctc tctctctcgc tctgtgtgtg 17100 tgtgtgtgtg tgtgtgtgtg tgtgtatcta cctgtagata gatagatatt 17150 aatataattt tagatagatg tatctagatc tctctctctc atatatatgt 17200 gtgtgtgtat atatctatat ctatatctat atatatctcc ttttaccctt 17250 aaatattcag tgtatatttc ctaacaacaa ggtgatttaa aaatatatat 17300 ataaacatag tataattaac aatcaggaca tcaacattga aacatttctg 17350 ctatgtcatc tacaggcctt aggaagactt tgtcaggtgc cccaataata 17400 gccttgatgg tagaagaaaa ccatgtgttg tattcagttg tcatgtctct 17450 tagtgtcttg taatctgaaa taattcccaa gccctttgga tttcatgaca 17500 gtgacattgt tgaagagtac aggccagtta ttttgtagaa ggtctctcag 17550 tttaggtctg tctgatgttt cctcctgatc agattcaggt tattcacttt 17600 tgacaggaat accactgaaa tgatgctgag ttcttctcag tgtaacgaga 17650 tctagagaca cacactgtca gtttgttcct tattggcagt gtgaaccttg 17700 aggatttcat tgtagtggca tttggcatta ctccattata gttactattt 17750 taccatttta aattaaaact atctggccgg gcgtagtagc tcatgtctgt 17800 aatcccagca ctttaggagg ctgaggcggg caaattgctt gaggtcagaa 17850 gtttgaaacc atcctagcca acataacatg gtgaaacgcc atctctataa 17900 aaaatacaaa aaattagcct ggcgtggtgg cgcatttgta gttccagcta 17950 ctcaggaggc tgaggcacaa ggcttgcttg agcctgggag gcggaggttg 18000 cagtgagctg aaatcacgcc actgcactct agccagggtg acagagtgag 18050 actctgtctc aaaaaaaaaaa agtaaataaa taaaaaaatt ttttaagtat 18100 cttatgggca tatacttgtc ctgttactcc tcaaactttc atccactttt 18150 ttttttttaa attttttttc ttacctttca tcgttttctt gatatccact 18200 gggttttagc atctacaaat gattcttgcc tgaatcagtt attatggtag 18250 ttgatggttt tctaattcca ttattccttc tatgtttgtt aattttggca 18300 ttcttctata aggaagagct tacccttttt ccctattaat taattcatat 18350 attaatgcag acctatgcat tcttacttca ttaaatcata atcctttact 18400 atcattatgt attctgatgt tcagactatc ccagattag ccaataagat 18450 ccccttcagg ggaatggtct ttgggattcc tctttagagg ttcctggttc 18500 ctgttttctt ttgacatatc ctattactct ttgagcattt tttttttttt 18550 ttttactttt aggcacagca agaagttcca tggtcctctt gttctttccc 18600 caactcagcc ctagagtcag tcacttctcc aatgagctct agttcctttt 18650 agtagagaat cataattaga aaacaagaat cagtgccaag tgtgcacctt 18700 tgtttttaag gtccatccac gttgccgtgt atatgtccag catgttgatt 18750 ctaactgctg aataatacct catgattgtc atccatccca gtgtttcttt 18800 ttcccttctg taatgaggga ctcctggact gcctccagca ttaccttcac 18850 aaatattgct gtgaggaaaa tccttaaacg tttcctttat gggcaacgtg 18900 tgagcatgtt tatgttgatt caggggtgcc agacacagct ccagaatggc 18950 tgcctcagtt tacatttcca ccagcagagc atgacaggct ctgtgtctcc 19000 gtgaataatc agcattaacc agcttcctat tttttgccaa actaatagat 19050 gtgctaggat aactctttgt tttaacttgt ttttctctga ttaccaatga 19100 gctggagcat ttcttcatat gcctgatggt ctttgggatt cctcttaggt 19150 aaattgctta ttcattataa tcctttgcct gtttttcact ggagttctta 19200 tatttttctt gaagatatgc aggaattcct tatacatcct agatattaat 19250 cccttcctgg tctcagacat tgcagatatc ttctgaatct gttattatact 19300 tatttattta caattttttt tttaagagtt ggggttttgc tctgtcaccc 19350 agactggagt gcagtggtat gatcatgact cattgtggcc tcgcaatcct 19400 gggcttaagc gatcctccca cctcagcctc ctgagtagtt gggactacag 19450 gtatgcacca ccagacttgg ctaattttat tttattttt agagatggaa 19500 gtcttaatat gttgctcagg ccaatcttga actcctggcc tcaagcaatc 19550 tttccacctc agcctcctgc atctattata tatatgttca ctttgctcat 19600 gctgtatttt gttgcaacat aaaactattt ttcccattgt tttgtgcagt 19650 ctctcaccag cactcttctt tttctgtaac tgtgttaatg ccctttgttc 19700 ttccatatgt taggtatgct ggtatagttg aactctgctg actctcctca 19750 gtaaacagtc tctttttatg acaccttatc ctctactgaa ttctctctat 19800 caagaatgac ttggccgggc atgggggctc atgcctgtaa tcccagcatt 19850 ctgggaggcc gaggtgggca gatcacccga ggtcagaagt tcaagaccag 19900 cccggccaac acggtgaaac cctgtctcta tgaaaataca aaaatcagct 19950 gggcgtggtg gcaggtgcct gtaatcccag ctacttggga ggctgaggcg 20000 ggagaatcac ttgaacctga gggggaggtt gcagtaagcc gggatggcac 20050 attgcactcc agactgggtg atggagaaac tccatctcag ggggaaaaaa 20100 aaaaaaaaaa aaagaatgac ttgtcttcct cttagagtgt gaggtctaca 20150 tacaaatatt attcttgtat tcagcaaatg tatgtcatag gcctagtgtg 20200 tgttaggaac tgtgctgtca ccaacaaagt ttagagaggt tataaaactt 20250 gactgtagct ttttagaggt ggaggagtga tttgaaacct aggctgtaat 20300 tccttcctcc tgtgattcct tcctactgtg ttgccttccc ttgaaaattg 20350 catttggggg ccaggtgtgg tggctctcgc ctgtaatccc agcactttgg 20400 gaggctgagg cgggtggatc acctgaggtc aggagttcaa gaccagcctg 20450 gccaacatgg cgaaaccccg tctttactaa aaatacaaaa attagctgga 20500 tgtggtgtgt ggtgacatgc acctatattc ccaggtactc agtaggctga 20550 ggcaagagaa tcacttgaac ccaggaggca gaggctgcag tgagctgaaa 20600 ttgcaccact gcactccagc ctgagtgaca gagtgagact ctgtctcaaa 20650 aaaaaaaaaa agaaaagaaa gaaaattgca tttagttcct gtagactgtg 20700 tgtcaaatgt ctaaatctct tctaaacaaat ggcctaagga ggtgcaaagc 20750 gaagcatcct caccagcatc ctgacttggc agtgaggcat gggaccctgg 20800 agggagtagt ggtaagtgtg actctggaat tcttcctggg ctacttgtca 20850 gtgactggct ccagattgag aggagagccc agaggacaca ggtggctgcc 20900 ccagcctgga ggtgaaagtc ttaaaataaa atgccagatg cctagaccat 20950 tctaaacctt tctgagaagc tgaaatcatc ccttctggaa gcgctctagt 21000 tctaaaagga cagatataca gcaagatctt cctggggcta atatggagtt 21050 tataggcaag taggcctcag aacctttccc tggtagtgat atctgtgggc 21100 aggcacagtt tccacacttt ccagaaattc cagcggaagg agtgagaagg 21150 aggaatctgc ccttgagtga ggaccaaaga aagcagaaat tcctcttggg 21200 aatttttcct ccagagacca aacactactt gggagcttgt ttactgggct 21250 ttaaaagctt gtgaccccca gtcactcttt cttgaccccca aggctttgca 21300 tttctgtggc ttccccactg gacagaagtg gaactgtcat gctgcctgtt 21350 ctggggtctc ccagaggttt ccccatgtcc tctccttgct tctactgccc 21400 cacagaattg gggatctgtg accacatatg gtatagaatt aatgcttgag 21450 aatggtttag ttcagtgatg tcaaataaga ttcactttta tgccacctcc 21500 atcagttgaa ggcccccctg gcccctaaat tggaaaaagat tctgagacag 21550 aatccccgtg ggtacagcgc agggacagta aaggcacgtg tgctgtgatt 21600 tgctatccac tgtgtggatg catccaggaa tatcagaacc ctggaagatt 21650 atttaagggg aagttaggac agcttttttg ccaatccaag ggtgttcttg 21700 aggaagtctg tcttcctgta tggccttcag tttctttcct gtgtaaccat 21750 ggggccaaca cataattccc acagctctat tggcccttgt ctgccaggat 21800 tctctagggt ctgattcgag gtggatcctg gccctttgag gtggcagaat 21850 ctgatcatgg tgctgtttcc ttagatttag gccttgatac ccttggcgag 21900 agcatcctgg gctgagtgac cacctgaggt ttttctggtg attttgtgac 21950 ccatgtaaaa ctttgagctt tgggattatt ctctcaagga aatagtgaca 22000 tttggtgaag agcctgtttg gtgtggctat gtgaggctta gccaagaaaa 22050 tgcaccattt ttattaggag gttaggccat ccgttgccac aaagtgtcag 22100 atgctaggcc tagagcctgg agaaaactta ttttaaaatt gatggggtgc 22150 tggagggggtt ggggggtggt ggctgtagct catgaatcag gtgctaaaacc 22200 tagaaacaaa aggcctcatg tggcagactg tttctgagca cagatgaatg 22250 gatgagcaac tggcgcaact ttgcccagtt ggtccagctt cccacttggc 22300 cacctaggct tgctgtgaag acctcgtctg gcagaaatga gagtgttttt 22350 gccccatctt gatcttaact gtaatttaag actaaaatct tagattctaa 22400 aacatcaaag gcaagatggc tcccagctct gtgagctcag cttctcacct 22450 cttagttgaa caagtgcagt gtgggtcaat acatgattgc tgctcttgct 22500 gccaggaact gtcccagcat agaaaggaat gggacacaat ccctgccgtc 22550 aagattctaa gggaggaagc aggcaggtcg actggtgcct catctctgca 22600 gggctccagc caaggtttgt gaaggatttt gcaggcatat ggagtgggga 22650 ctgattgatc ccgagagggg actggggaaa gctctgaaga ggggatgaca 22700 tttggtttga actccaaaaa atggttgctt tacctgtttc ctgaagtttt 22750 tgaggtggct tataagaaca tataccataa aaaggaccaa tataaattta 22800 aaatcagaaa aagagaaaat gggctgggca tggtggctca tgcctgtaat 22850 cccagcactt tgggaggcca aggtgggtgg atcgtgaggt caggagatcg 22900 agaccatcct gcctggccaa catggtgaaa ccccggctct actaaaaata 22950 caaaaaatta gctgggtgtg gtggcacatg cctgtagtcc cacctacttg 23000 ggaggctgag gcaggagaat cgcttgaaac ctgggaggcg gaggttgcag 23050 tgagctgaga tcgcaccact gcactccagc ctgggcgaca gagtgagact 23100 cctcctcaaa aataaataaa taaagagaaa atggaactta gaaaattaag 23150 aggaagagtg aaaaggtaga tatttagtca ggcacagtgg ctcatgcctg 23200 taatcccaac actttggggag gccaagacag gaaaatctct tgagaccagg 23250 agcttgagac ttgcctggca acatctcagg tgagacctta tctctacaaa 23300 aaaatttaaaa attagctgag ctgtgtggct cgtgactgtg atcccagcta 23350 ctcaggaggc cgagaccaca gcccaggagg atcgcttggg cccagcagtt 23400 tgaggctgca gtgagctggc accactgcaa ttcagcctgg gctacagagc 23450 aagacccagt ttaaaaaaaaa aaaaaaagat attcaaacca tgggtcccaa 23500 cgtagttat atatttgacc atttgcaaaa gctgaaagca aaacatgtta 23550 cacattttca gagaggaaaa tacacagtag ttcctgagtg taagttgttt 23600 ttcttgacct cattcttaaa ttgcttcatg agggtgggag ggaagtggta 23650 gttaataagt gaacctgtaa accagcgttt ctcaaaatgt agtccaggga 23700 attgcatcaa aattgcagtt acctacagtg cttgttaaaa tgcagattcc 23750 tgggcccctg ccccaggctt atcaaatcaa tctggtgagt aggactcaag 23800 aacctgtaaa ttcacatact tctgcagatg attcttcttg cactgcacag 23850 catgaaagcc tctgcaatag acagaaagct accagcattg cgaaagcaac 23900 ttgagtgctt ggcctttgaa ggttgagtgg gactttaatg agggagagag 23950 taaggcatga gaaatggcag ttccactgag gtcagtcagt ggttcattgc 24000 tgacgaagtc acttttaagt catgttttag aagaactacc aagtgtggca 24050 ggtcaggcat gtggcaggac tgtttctgag cacagatgaa tggatgagca 24100 cctggcccca ctgtgcccag ttggtctagc ttcccacttg gccacctacg 24150 gtctgctgtg tggaccttgt ctggcagtct cctttaattt attttttatt 24200 atttttttct ttttgagatg gagtcttgct ttgttgccca ggctagagtg 24250 cagtggcatg atctcggctc actgcagcct ccacttccca ggttccagcg 24300 attctcctgc ctcagcctcc caggtagctg ggatcacagg caagtgccac 24350 cacgcccagc taatttttgt atttttaata gagacatggt tttaccatgt 24400 tggccaggct ggtctcgaac tcctgacctc aggtgatcca cccatctcag 24450 cctcccaaaa tgctggaatt acaggtgtga gccaccgcac ctggcctatt 24500 ttttttcagc aaattctttg tttttctctc tgttcccaaa tgcagggtac 24550 tgagaccaca gatgtattct gtttcctgtt gaaaaaatgt ttctcactta 24600 gctgggtgtg gtagcatgca ctgcagtccc acgggaggct gaggcgagag 24650 gattgcttga gcccaggagt tcgataatca tgccattgca ctctggtctg 24700 ggtaacagag cgagaaactg tctcttaaaa aaaagaaaaa gaaaaagagg 24750 tcctagggaa agaaacaaaat agtggcttgg atggtgagtt ggtggaaaga 24800 acagtgggtg ttgggggtgt tgaacttgtg tttgtgtgtg gtgtacccaa 24850 gacatatcat gtcagcatta agaatagact attcctgttt tctggtcact 24900 gagttgtatg ttttgacatc cttattttgg aagatacttc cttactagga 24950 atgggatagg gagggggtca cctttcccat ctgtgggtca tattttaaaa 25000 tatttattgt tcaagtttaa agatataacc aaaggtataa agaaaaatac 25050 cacaaacatc tgatttaaga aacaaaccag ccgagcgcgg tggctcgtgc 25100 ctgtaatccc agcactgtgg gaggccgagg caggcagatc atgaggtcaa 25150 gagatcgaga ccatcctggc caacatggtg aaaccccgtc tctactgaaa 25200 atacaaaaat taactggtca tggtggtgtg tgcctgtagt cccagctact 25250 cgggaggctg tggcaggaga atcgcttgaa cccaggaggc ggaggttgta 25300 gtgagccaag attgtgccac tgcattctag cctggcgaca gagtgagact 25350 ccgtctcaaa aagaaaaaaa aaagaaagaa atcatttcct acaccttcga 25400 agccttcatg agttagattt tgaaacagtg caaaatgctt cacgtgagaa 25450 tcgagagtcc cttctggtgg ctctccatcc cctgctcttc tgtcaggttt 25500 tcttgtaggt ttatggaaac ctttgttact tgtgcaggtg gcagagaagc 25550 agagaggata gctgcgcgcc acccacacag ctaggattta ttggcgtact 25600 cccacgtgca tggcagccaa gtggacacaa ctctgtgatg aatcctccca 25650 agagaactga ggggccctga tggaggagct gcttctttgc aaagctttcc 25700 ttgactctct tcctgtcccc tagttgattc cccttctgtg ctagttttag 25750 cttattgttt gttacctgtc acacttagca gtactgttgg ctttgctggt 25800 ctccttgact actgggggta aagacctttt gttgttgttg ttgagacaga 25850 gtcttgctct gtcgcccagg ctggagtgca atggcgtgat ttcggctcac 25900 tgcaaccttc acctcccagg ttcaagagat tctcctgcct cagcctccta 25950 agtagctggg attacagcta caccacaccc ggttaatttt tgtattttta 26000 atagagatgg ggtttagtag agatggggtt tcaccatgtt ggccaggctg 26050 gtctcaagcc cctgacctca aggtgacctg cctgtctcag cctcccaaag 26100 tgctgggatt acagacatga gccaccatgc ccagcctcaa agacctcttc 26150 tttacttgct caccctgccg cccactcccc taccaaccct tgcatgccct 26200 ataccacctg gcacatgata catactaact gggtacatgt ttgaatatga 26250 atggatgtgg tgctgtgaat gcttagggga agtgggtgaa atgcttaaga 26300 accaaccttg agtggtctgg gaaggcttcc tgggagggtg gtgtttgagc 26350 taaggccagg cagctgttag atttgttaga ctgaagccct tgcagactta 26400 gagagcttgt gctcttccca gaatgacggg tgagccacgt acagtaaatg 26450 gtgcttctca tttctagccc aagggggcctc aagggggcacc gtgatttcac 26500 gagaatgctg caagcaaatc ttttctcaag ctggggaatt tggtggtaat 26550 gcctggctca gcttgcggtg cgcacctggc ctttggaaga ttggtacaga 26600 gagaagcggc ccatccacat gagcctgtgg aacagcactg gtgggggagc 26650 tgatttgtga agaggggctg tgcagtgtac tgtcaggtct gagacccagg 26700 aagaaattcc agtatcccag ctctcagaat cacagagttc taggcactgc 26750 ctagttccac gtgttcccaa atgtttcctg aatacttgga tttcctgtcc 26800 agagaatttt caaaacaaac ttagaggcct gacccatggc tgccaaggaa 26850 ggattttttt tttaaattaa attttaaaaa tcagtccagc atgaaaatct 26900 atgatgattt catagagaa aggacatttt aatattcaaa gagtaagaag 26950 cacttaatct tggaagaaag ggcattccta tactttgatt acctttagtt 27000 taattaaaaa acacctacat ggtctttact tctgtgattt cattcctggg 27050 ctagtgaaac attgtcacaa taaagcatca ggccaacgct tctttcgacc 27100 cactggccaa tcagttgaca aacagtgact agatgtttca gcctattttg 27150 ctgaggctaa aggattgaac tagtgcttca gccagcatga aaaccagtca 27200 ggagtccgtg ctggtgttgg cttagattag cagggccttt gatgggagggg 27250 catgtatgtg tttgggtttg ctgtgccagg caggggagca gtggaatttg 27300 tctgaattga gctcacacat tgaagttatt gagcgactta catgcaaggc 27350 catgacctgg actcccagcc gagaggccca cgtggcgggg cttgagctgg 27400 gggagccgag gacagcttac atctgctcat ctgcttacgt aaccctgcct 27450 cccagcttcc agagccaaga aaaacacacaa gccagcccag cggggccgag 27500 agcctgtggt agcacacgcc atgcgccgca cagcaagggc gccttggctc 27550 ggcttgaggc ctgtcatgaa gccctcagcc ctctgcctcc tcccagagct 27600 tctccccacc accccaggca gtggctctga aacctggtcg caggtctgca 27650 tgattctgaa cagaggtagt cgttgccttc ctggagtctg agctctctgg 27700 agtttctcac tgggacagag ccaggtgtgt agcagagcat ggtccctgca 27750 gtatggcagg aggtgtgcag ggcattcagg aggcctcctg gctggcactc 27800 gacccaatta gtcattcaac gccaggtctg gggctgctgt ctgttgtctc 27850 aaaggtgtga gctgcaagat ccttagagtt gtggagaaaa aattgccaga 27900 ttggcaagaa gggcaggatt gggggtcaag gtgtctcagt gtgttggaag 27950 catgatgggg gttgtgcaag gggcacagcg agttcagaag ggagcaggag 28000 agtgagaaga ggctgttcag tgataaagct ctgcacagag ccattggagg 28050 agcaagctcc ttgaccatcc ttaaaccagg gtaattttca tttaggttct 28100 gccacacgct cagcagggaa ctcctggaag gcaggatttg tcttgtccat 28150 cctccctccc tacctcaacc cactcctcct tgggctggca cacagtaggt 28200 acccaaag tatcaattga aacaaattga aagtggtctt gatacatatc 28250 acagggcaag tttgcagtta acagacattt cagagtaaag actctctggc 28300 ttggtgctcg atcggcttct gtgggttgtc agcatgctgt ggacagcccc 28350 ggcatgggag cgagtgggcg tgtgtgtgtg tgtatgtgag ggtgagagag 28400 cgttagtgtg tgtgttgggg ttggggagag aggagggggga atagaagatg 28450 gaccacccgg gtatcagctt ctgccctggg gagatggtgg tgtcagttgc 28500 tgagggaatc ctgagaagca ggtctggctg taggtggtga tggtggtggg 28550 gttgcatgag aatccatttg gggcaggttg aatttgaggt gcccatgaca 28600 tatggctagc catgttctgt tggctgtgag gtcaggagag agacatgaga 28650 tggaaacaga ggtttgggaa ctgtcatgtg cttaaaccaa agacctgggt 28700 atagggagag tgagaagaga agggggcaaa gatggacatc caagaaagaa 28750 gctgagaaag cctaggaatt tgaggtaaga ggagacgtag gtaaatgtga 28800 cgcttggtga tcaaggcttc tttccacctc tcctatgctg gacactcacg 28850 tctcctgtct gcttggaaat tcatgctgag ggcagggaag gtgggagcaa 28900 ggatttgtct aaagatcttg ctttggatcc ctgcactcct cctggtttac 28950 caagtgtcac tggacacgtc agggcgttct gagaccttag agagcatcca 29000 gtcctgtccc tgcagtttac aaatgaggaa accagtaccc tgagagtggc 29050 tgtactatcc actctcagga taccaaagat catctggaaa gtcactggtg 29100 gagctggacc ggggcccagg catctcttct cctgtccggg gctcttgact 29150 tcaggaccac ctttctgaaa cccatgatgg ggcaacacca ggacactttc 29200 cagcctgcag gtgtctgtcc cgcggaagcg agccaggcca catgtgaatt 29250 cctgttttct gggtgggttt cagaaggtac gagcaagtcg gcagggtgac 29300 agcccaggtg cttcttgggt tccccaaac gcggttatgt ttagcagcat 29350 cctcagaacc aaaggtgggg tgggggctgc agatgttgtg ggggccctct 29400 gaagtgaaaa gagccctgtg acagatcttt tcttcatgtt tttcacaagt 29450 tcactgtgca gcagggcccc cccagtagcc tttgcccagg gttgggtgtt 29500 gggcagccca ggcctggctg accttgtggg gaagggtgtg aatggtggga 29550 atccccgagg gccctctttg cccgaaagcc ctaagccttg acatcagatg 29600 cccatcagat ggtccatcgg agccctacta cccagcttgc ccagtgagaa 29650 tcatctgggc tccttgttag gtagccattt aggtccttcc caaaatccac 29700 agactctcta agggaagggc ccgagatgct gtacttgtac taacttcctc 29750 aagcaattct tgtgataggt ttgggaaaaa cttgtccagg gtgaccactg 29800 actgagtcct ggtcttctct gaagagcaca gtgcctgctc actttagggc 29850 accctgggag gtgggagctg gctcagcagg cagtcttata agggactgag 29900 cttcaaggcc tctgtccctc caggagggag gtgcatgacc agagagggag 29950 gcctgaggat cttcttccct gccccagagg gtctgctgcc tgagctctgt 30000 gatagcgcag agagtaaaag gatcaagctt gattgaggcc tatctctcaa 30050 tgcgaaagtt tgctagttaa gaggagagtg ggaagggcat ttctggcaaa 30100 gagaaaagtg tggacaggca tggcttaagg gatggggagg gagacagaca 30150 gagctgaggg tgaagggcct tttgctcagc tgtgggcctt ggccttccct 30200 tgtgcaggga cacacagcct tagagccact ggaggtttta gtgggaaagt 30250 aatatggtcg gggctgtatc tcagaagaaa acaaactaat gggaacaggt 30300 cctgtgatgg tggacctggg tcagctacgg agggagggaa gatgtgagat 30350 gtgtactggg gaagggggtg gaagtggcag ctatctggtg agaggaagca 30400 ggcccacagc tttttttctc aagctgttga attcagaagg gcgagtgatt 30450 ccgggagtag ggggtgcttg gagagccacg cgttatgat aaacagggca 30500 ggctgaagcc tgctcactgg ccctgggcgg gttctcacca gcatgtttca 30550 ggttttgatc tgtgcttgtg gttggtgttc ctacctgttc tctaggttcc 30600 ttcctttgtt cttgtggctc atttgcttca caggtgaagc tggttacact 30650 agagtaacag ttcccaaagt gtgttccctg gaaaaaatggt tctgtagcca 30700 aataagcttg ggaaatggtg ggttaaatat aacgaagggg gtttttcgac 30750 tgcacaactt ctcagagcct ttggtgtgtg tcgtgacttt gcagaagcag 30800 gatttaatac gcagcattcc cgttcttatt tgaccacgag acatgttttt 30850 ccattaagca tcttgctggg tctgatgttt tctggaaccc attttgaggc 30900 ggtctggtct gcagagagta tggggagcct gggttcaagc cttggctctt 30950 gactctcagc agagccttga ttccctgtgt tgcctggact gcaccacgtg 31000 taccacatac ccggtatgtg acgttttcct catccctctt cccacctgcc 31050 gttacctcac aatccacaat ctgcacctca tccatttttc ttctgaggca 31100 agcactctct tactaactta cttatctcat ctgcatccat gttcttctag 31150 gccagaaact tggggagtcat ccctccctct ttgttacttc ttcttcctct 31200 ttgttacttt atcccctctg ttactaaaaca ttcttctgtg tttccagcta 31250 tttcttttat tttccctcgg tctcctttgg ggtttctttg cctccatctc 31300 tcccagacct tggttcacct tccatcgagt cccttcctgg gacatgggca 31350 ctcatgccac tcctgctacc ttccacttcg aagctaactc cctccacact 31400 gacgtcccca acatgcatgc atacacacac acacacacac acacacatac 31450 acacacacac acacacactt ccccagttag gctagaatca gagagatgat 31500 gtcagccatt tgtccaaggc cacgcagctg ggaggtcaca gagctaagtc 31550 tcaacctcag gggttttgag aaattgcctt ctcatccgtg atcactgatt 31600 tctacaacag cctgtcagga agtctgggta gaaattactt ccattttaca 31650 gtggagtcag agcggggagg gtcctgggca ggcgagtgct tcacagagtg 31700 accaaccatc taggtttgcc ccacactgaa gggggtttct ggggatggtt 31750 ggtcacccta atgctggatg tggtgcctga tgctgggcag gagggccctc 31800 tccgtggcca cgttgcctcc caggaggaga catttcctct gcagctgcag 31850 ctgcagcctg gccatctgat gcagcctgtg gagcggtggc gagtcctgtg 31900 gcctgctaac ttctccctcc ctccacctct ctagtgggcc ccatgctgat 31950 tgagtttaac atgcctgtgg acctggagct cgtggcaaag cagaacccaa 32000 atgtgaagat gggcggccgc tatgccccca gggactgcgt ctctcctcac 32050 aaggtggcca tcatcattcc attccgcaac cggcaggagc acctcaagta 32100 ctggctatat tatttgcacc cagtcctgca gcgccagcag ctggactatg 32150 gcatctatgt tatcaaccag gtgaggcctg ggaaggtgga atgagagagg 32200 gtgtgtgtgc atgcagatgt gtatcagatg tgtgtgtaat gagggcaggg 32250 gaagggggagt gatttcacag acacctggca cttacagcga ggaaccagcc 32300 ccccagccac caccagtgca gatgaggtaa acgccaaaaca gtgtgcttgc 32350 ctattgctgt caactctata gccaagggaa atgctggagt gttttcgttg 32400 ttctgttttt gttttctgga agtagccttc cagcaagatt gggaaaaaag 32450 acaaccctaa ttattccaaa gtacacactg attattccct ggctttgtgt 32500 agctgtgtat tttcctttta aaaataaaac caccatttag atgtcagact 32550 tttaggtaac ttcaaagttt atccagtcag tcagagcgtg tctcctgggg 32600 cacctggaga cagtgccctt agttcaggtc acatgcctac atgccagccc 32650 ctggtgaaat atctggagaa gtctgattcg tgggccatct gagagttatg 32700 tggactgggc cgagtctgag aaaaagtttc tcactgctcg tctgatccat 32750 atgtgttggg ctttagccct gcttaggaaa gtaatgctaa ggataggtca 32800 actttcatca ccatggcatg gagaatcaga ttgatctaag aggcatcttt 32850 attgaaataa atttttcagt ttatttgagg agcattattt tcccaagagt 32900 ataactttga tatttcaaga ttacccctaa cacttaaatt catgttttta 32950 gactataacc tcctaggtgc aatgacacat ctaacttatc taagcaccca 33000 gtttcattga aattcatttg aagagtctga gtacgcccat ttctacaagg 33050 cccaatgtcc atttcatttc gagataaact ctgctttagg taggaggatt 33100 gttggcagtt tacggcttcc atcaaggtca aggaactctg tgcaccttcc 33150 ctatgacccc aggggaagca ctcgaggact gctgtggcat tgtgctgcat 33200 cacttgctgc agggagattc tgaagaagtg taaggtctca gtcctgccct 33250 gtcccgaagc ctccaaccca cttctggcaa gtgggacctt cccagggaac 33300 aatttgttaa cagacccaaa tatcctgtga ttggatggtg gctgccaaat 33350 gctttggaag ctcagaggaa ggagagagag caatggcttg gaagaaccag 33400 gatataaact aggttctaaa gtctgcaggg agatgggctt ctcagctggg 33450 gccagtgagc agggacctta aggcagaaag gagccttgca tgttcctgga 33500 aattgagatg cccactgggg taggaaagca ccagaagctc tgggaccagg 33550 tgtcagagtt aagcctgtga ggcaggagag agcagaacaa gccctgttac 33600 aaggaaactg aagcaggaga gcaggtggtg ggcaaacccc ttgaggctgt 33650 ttgaattctt cggccaagtg aggtacagac cagggcccta tgaacacctg 33700 caagcaagac agccacgcag ttgtgggtca ccttggaaga atattggaga 33750 atgcaagaga gaacaggtaa atgtcctgca aaatgcgggt cactttaacc 33800 caacacatat tcatttaaga aaagctctgt gattgagaaa catttgtctg 33850 atgccagtta gcacatacca atgacggcaa gattcaggag cctgttatta 33900 aagcagtggc agcgagcacc tggaagaggc ggccaccatc accaggagcc 33950 agcagggatg actaataagc cgtgccagct gcatctcgtt tctctcttga 34000 cagttgctat gccagtagat gagggatgta ctgtggatac aatgctgtca 34050 tatcttattc agcagggcat ctgatagcat ccccaaaatc tgcctgagta 34100 gaagacagac agctgtggtc tgggtgccat ataggtaggt taaaatatat 34150 atttgggcct aggcgcagtg gctcatgcct gtaatcccag cactttggga 34200 ggccaaggca ggcggatcac ttgaagtcag gagttcaaga ccagcctggc 34250 caacatggcg aaaccccgtc tctactaaaa atacaaaaat tagctggaca 34300 tagtggtggg cggctgtaat cccagctact cgggaggctg aggcaggaga 34350 atctcttgaa cccagggaggc agaggttgca gtgagccgag atcatgccac 34400 tgcactccag cctgggcaac agagtgagac tctgtctcaa aaaaataaaa 34450 taaataaata aataaataaa atatatactt gggtaaagag gataaaagag 34500 ttagcgatga tgctgaattt ttgaactgag gtggctgttt tcaaggaaga 34550 ctggagggtg ggatgctacg tctagatatg ttgcagttta ggtgaatgtg 34600 agacttccct gttttgaagt caaatattgg accagtaaaa tctagccatc 34650 agcttaaatt cctatgatac aatttacata ctccccaggc tcaacacagt 34700 agatttctga atgtcctctg ccagctacat gctcctgccc acctcaatcc 34750 gagtagatgg aacaactaac caagccagct cagaccggtg gcacagctgt 34800 gctggctaac actgggcacc acctaagaga gtgcttctcc aaaagtgtgc 34850 ttccccaaat ggagcgaaat acgcttgagg aatgttgggt tgaaccatgt 34900 aaagcaggtc tcattcccgc agagcctttg gtaccccggt gtacactgta 34950 accccagaag tgtttcctga gcttgcctga cgagacaact tttccaagaa 35000 ccgtctcaag tgatgagtgt tttgtgagtc acactttggg gaaagcgggc 35050 ctaagttagc atctcctccc agctgcctcc ctgctttccc tggaacacta 35100 ggaactgccc gtcctccctc cctccctcct cttcccactt cacaacttag 35150 catcaggaat attttagttt tggtttttca aacatatata cctccttttt 35200 tcttatcttg tcaatatcat ctttttttt tctttgcttt tcctcatact 35250 tttttttctc ttcatccttt ccttctccaa gggttaactt tccaccttag 35300 gagaatcttt tctgcttttt ctcccacttc cccagctact ctcttatcat 35350 ctgctccaat ctcaccctaa ttgatcattt tgggaaaata tggtcagagt 35400 ccagataact aagttgagaa atgcttaaac tctgccatac ctttccagta 35450 aagaatatta cctaataaat aataaaatgg taatgggaaa cctgaaccct 35500 gaaaaaaaag aggtggaagg agaaacatt ggagcacatc ctgtctacaa 35550 attaggaact gcctgtgtta tctgttttat ggttatattc tagaagaaga 35600 aagggatttt gtagcacctg gttttgacct ttctgcactg tttgttgagc 35650 aaataaacct tatgggctgt tagccctctt tatagcctct cagcttatcc 35700 ctggcccaga caccctgctg tcattttgac ttttcattcc cacacacaca 35750 tacacatgca cacacatgta cacacacaca cataccattt aagattagac 35800 agaagtaatg ctcaaaatgg agtggcttct gagacattta gtccaagggt 35850 tcccaaacag gcttttcagt atcagatttc tttctgcccc attgaaatgc 35900 tacacaacct tccgcttaca gcaggtcaca agggtttcat tctacttgaa 35950 gtaggggcca tgtcccattt ccacttcctt ggcttcccat tcagtcactg 36000 ctaggatttg cctagacccc tgaggccaga caatgtagaa acttctgctc 36050 catgtcacag gtgaggaaac aggctcagag agggacaggc tccgaaagtc 36100 acatagacaa cagtagggct gcggctcaaa ccccagcgtc tgactccagg 36150 tttagtgcct tctcagggca tcagtgacac tcctcatggc cagggtgccc 36200 ccagtgttgc tcacagtctg gtatccaggg ctgagagtgt gctgtgtgct 36250 cagactgcct gggttcagtc ctggcactgc cactttacag tcagtgacct 36300 caggcaggtt acttaagctc tgcaggcctc agtttcctcc ttggtgggga 36350 gggttatgag gcatccttct catggtaaac cttcagtaaa taccagccgt 36400 tactaggagg gtccactcct gcctctccac tctccattca tcctgcctgt 36450 ttcctctgcc tgcttcctct gcctgcttct gtggtggtga attcttcatg 36500 gctcccaccg cctcctgctg cacccccact cagggcccgc atcaggaccc 36550 ttcctcctat tggtttgaac tccttggagt cagagggtaa tggatagtgg 36600 agtgagccag gtggcagaat ctcagaggcc atcccgggcc tataagcctc 36650 ttcaaaatag ggccacgtat caagctttac acacaggagt gaactttcac 36700 aagttgttat gactcatact ctgtctatag taagctgtta accactccca 36750 tttggcttat gcctctgtaa ttattgtact aacttatatc ttaaaataag 36800 gatattgaag gaatgagccg ggagaggctt tcctggttga gatatagaag 36850 aacaagagtt gctctttttc cttaaggtct ctcctcccac ccctgacctt 36900 agctcaccag catgggagaa tactatttga ctccttgtac tctgagacgt 36950 ggatttcaag atatagcatt ccaacttcaa cggcagcaag aaaagaagca 37000 acagaaggag aagacatcat agcaaacagg gatgcatgct gcatttccta 37050 atactcaaac ccggaaacga gacttcactc aaggtgaagg gagggcaggt 37100 caccacctgg tagcactagc cctaaattaa ggaatgcaga atgtttgtgg 37150 gattgcccat cataaaaatt acaaaatgag taaggaatgc aggcacagct 37200 ggccaggtgg gtttgtcaca accatggcag ccctttgccc cacagccagt 37250 acacagaact ggtctctcca attccgattg catatcttct ggcacctctg 37300 ttcctctccc tcagctgccc aggatttttc tggttctgac catgttactt 37350 cctcttttaa acctgttagc atttcacgac tgcctacagg caacggtcta 37400 aatggtcgga aggcccaagc ttagcatccg agaccctgac ctacctccag 37450 ccacttcctc ctcctctcca cttcactgga ctccccatct ccacccagac 37500 acctctgttc tcccctctgt gtgcctttgc ttatgctgtc ccctgtgttc 37550 ctagtgtgtc tctggctatc ttttaagctt ccctccccaa cctcattagt 37600 tctgtggagc ccctggaata gagctgactt ctccttccct gctgctccca 37650 ggctgctcag aactttctgg aaaggggatga ttatctgagt tccagcctca 37700 ccccagcccc cggactctga gtccctcatg tctgcctccc ttctttctct 37750 ctgaccacac agctggtaca tagtcagtac agacgcagtc agtgagtgga 37800 gcacggggct tctctccagg attcctgccc ctttgtttat ccctagtctc 37850 aggactccct actcctggtc ttctgcctaa atctgtgcct cttggaagtg 37900 aagcctccgt tcccagtggg gccaggtcct gacccttggg aacttgcagg 37950 atccctccct tgggcctctc cccgaagctt ccagctcaat gctgaccaga 38000 gcacaggctg cctgtgacag tccttggggt gacctccctt atcaggaaaa 38050 atgcagaaaa cctattaata ccttagcctt gtgattgtta atggtcacaa 38100 aactccttta gggtcctttg gactcagcac ctttatggtc tcactttgaa 38150 ttttgaacct cccacctccc cccatccccc agagtaaggc aaatggtctt 38200 ctgattgttc ctgcagaggg aaggctccac aggtaagcac acgatggcca 38250 ggaagcagag ctggagcctg cctgaaaggc tgtggagaaa tggaggggagg 38300 gctgccctga ggactctgtc tggctttgaa gttttctact gtttcctttt 38350 cttctgtgca ctgttttagg atgatggggt gatagttcca ggctggttga 38400 ggatggattt ggagacagtc ctttgtaccc tcagtgagca agagtatctg 38450 tcaccctacc tcagcagttg tctctgtcac tggtccaagc agctggttcc 38500 tacacaaggt caagatcaac tggggagaag cagactcctg ggtctatccc 38550 attagtgagg acagctgcct gggcttatgg cctcattggt ttggtttcta 38600 tcttgatcat ctctaccatc cccccatccc ggccttccat tttctacctc 38650 agctgtcagt gcacagattg atgtgtgtgg gaacggagct tgggaggagt 38700 ggggtagggc tggtcctgtc ctgtagcctc cccttccttc gggcacttgg 38750 accctttgga gcttgccggg gtggggaatg ggagtgggaa ggccagggag 38800 tgtctctgca ccatcactgt ttgagtgttg cccctttgct gtgtgcccca 38850 cctagtctat gtgtgtctct gttctctggg gactcaattt gctggtgaat 38900 tgcttccatg gacattgttc tgggaaatgc cattttttct gctcacccat 38950 gactctgtga caaggaatga cagcttatta ggaatttgtt tttgcattgg 39000 aacagtggtc atcagaatgg gccccttttc ccttgcagct ttgacatttg 39050 cctctctttt cctcacctct ctcccttgca tccacccttt tctctttttc 39100 ttcttttttg ttttccttct agcaggggcc ttttaccttt acttgttaat 39150 cctgtttgta gcaaagcaag tggaagggagg agttcctctc tgatctgctt 39200 cttatctcc acctaccttc tcttctgtac tttccgcctc ctagagagag 39250 agagagagag aggaatgccg acctaactac cgctgccact gctgctgcca 39300 ccaccgctgc caccaaccacc ctggtaatgt tcacatgtcc tcaaatcaac 39350 ccagagccag ggccctgctg gtcaggggga ggctatgtaa ataatcccat 39400 gagtgtgcca tcctcaggcc ctggggtctc ctaggcaaga ccagggcctc 39450 tgtgggctct ctcggaaatg ctgaggttgc tggaagccag cccgtcatac 39500 agggtctgag agtttaactt cttttaaatt aaaccacagt tgagctcatg 39550 ctgtgtgtgt ataaactttt gtatcctgct ttttccttaa attctttatc 39600 atcagcatct tcccatgtta tttcatagtc ttcatcatca tcactttcca 39650 taccttcata gtagttgatc gtagaattcc atcataatta acttgtcttt 39700 tctctcttag aagtccctta ggtaatgtcc aattttccgt gagtgtaagt 39750 aataccataa tgaacatctt ggagtctgaa gtttatctg tgttggtttg 39800 ttccacatt aggatcattt tcccaggcta gattttcaga tgtgggatta 39850 tgggttcaga tatggtttac acatttttat agttcttaat acagatggcc 39900 aaattgcttt ctgaaagaga agcttttctt aagtattttt ctccaacttg 39950 tatcttaaac atcctgaaca tgcttagcac cactgtcttg atatatctgc 40000 ggaaagccac gtctccactt ttcagtgtgt cgggccctgg gagaggcagg 40050 catcctgcgc tggctccttg gagctgggtt taaaattgtc tcctctggct 40100 gggcgtggtg gctcacacct gtaatcccag tactttggga ggccgaggtg 40150 ggcggatcac taggtcagga gatcgagacc atcctggcta acatggtgaa 40200 accccgtctc tactaaaaat acaaaaaatt agccgggcgt ggtggcgggc 40250 acttgaaaag tcccagctac tcgggaggct gaggcaggag aatgatatga 40300 acccgggagg cggagcttgc agtgagccga gatcgcgcca ctgcactcca 40350 gcctgggcga cagagtgaga ctccatttta aaaaaacaaa caaacaaaac 40400 aaaaaaaacaa acaaaacaaaa actgtctctt ctgtgctcac ttcacccaga 40450 atccctgttg ggctcttcaa ggagctcagt tctctctgaa agcaacttta 40500 tagcctcagt ccagtctgtg ttcctgtgtg gcaggggtca agggtatgct 40550 cactcttgag agtggtgtct ttggttgacc aagaaccact cccatagcct 40600 ggtccctaac ccttgaaggc ccatctctct cactcactgg ggtgaagagt 40650 ttaaatctca gatccaagtt ttgttgagag ctctgagcta ccatattgct 40700 atggttaaca atagttaaca atgttaacaa tggttaacta tggttaacaa 40750 tagttaacaa tgtttaacaa ctagagccca gctgggtgtg gtggcatgtg 40800 ctaacagtcc cagcttctca agaggctgag gtgagaagat tgctggagtc 40850 caggagctca aggccagcct gggcaacatg gcgagaccct gtctcccctg 40900 caaaaaaaca acaacaaacaa aagcaaaact agagcccaac tgctgtgaac 40950 tcatggctga gtagatatta ttagccctcc acaaactcag catttgtata 41000 atcccaggct gtttccagta attctctggg gatcatctcc cagcctgtcc 41050 actgttccag gatccacact taggcctata ggaatgcccc gtcagagctt 41100 ctgctgccgc tgatctgtta ctgtttcatg caacccactc ggcctagttc 41150 cttcctctta ctgtctcagt gggcacagaa aagcatacag agggtgtttc 41200 agcaaacatt gccactggct gcagacctgc ccccggatct gtcctgttga 41250 gagcttagtg ctgcgttctt gcatggtggg gaggggtgtg gctctgtgat 41300 gagccagggc atgtgtatag gagcaacagt gtctctctta tcacgtagaa 41350 gttctgactc attgcgagtc ttggctttgg gttaatggtt ccagccatgt 41400 tgctgctgtg tcttttggtg caggagaggc tgggcacagt tggtccctaa 41450 gccattatgg ataagggatg tgtctgctga tatacacaca tggacctgac 41500 atccagggaa ggcagggtga ttggacagaa cagttcttcc agaagctgtt 41550 ggaacttgga caagagtggc ccttggcttt ctgtagttgg tcatctgtcc 41600 cctgttgcaa tcaggggaag gccacacttg ccttccttaa ccacagttag 41650 gattttcttg gggattagac cagattctag cacctgtcct gaacctctcg 41700 ccccgcccct acaaaggctg cttgcaagtg tagtgcacat acacagggag 41750 caggtggggc atggaagtgg aagtggagcc cctgcctttg gcccttgggg 41800 gaggcactgt ctgcttaccc acggttgttg cctcatagga atcatacaac 41850 agcttcctaa ctggtctcct tgccttcagt tggattgggg cacaaatccc 41900 tccttgacat ataaaccatg gtttaaggct ccctgtggcc taaataaaga 41950 taaagcttaa gtatcttaac aagcacctaa cccttctccc cagcctcggt 42000 gatttggctc atcgctgcct tcatgtttca ttctggcttc actcattcgg 42050 aatttcttgt agttccttgg ctgttctctt ttccttaccg cctttacaaa 42100 tgctctcacc atgcatgctt ttctctgctc ctacagatgc cttctctccc 42150 agcaccgcct ccagagtcta tgtctggtcg attctgtctg ctgtctccag 42200 tccccatctt gtggcagtct ctgctcaatc atttggggat tttatatgtt 42250 ttctggcctt tcttttgggg gcctgtcttc tccttctaaa agcagccagt 42300 tgacctagaa ggaagggata actgtaactc ttgtctacca acataagatt 42350 aggcccacc tttaaaagct gcgtctttga aagggacacc tgcacccagc 42400 atgctggctt ctcttcacca agcgtgactt cctacgcatt tcacaggcct 42450 ccagaggtcc ccctgactct cttctgctgt gagaaactct aatcatgtaa 42500 gccacaggct aattcccttg agccttaaat gtttttagta atttcccatt 42550 catcagagaa gcaggatttg ggaggaattt tgaagcaaac actacagaag 42600 gcagagtctc caggtaggat atctaagaga catttggaat ggtctgactg 42650 ttcaagatgg atgggaaagc ctcttcctgt aatgatagta gccaaacattt 42700 gttgtcaggc agtggggccc catttttgag atggggtctc tgtcacccag 42750 gttggagtgc ggtggtgctg tcatggctca ctgcaacctc agcctccccg 42800 ggctgggtct tcttaattct gaaaaaccca gcttttaaag ggtggaccta 42850 atcttatgtt ggtagacaat gttgtctcat ttaatacaat gcacatgctc 42900 tccccataac acaaaagagg gaactgaggc ctggaggtgt gatgtacccc 42950 aagtcacata gctaataaat aaagaagcca gcattcctgg gattaaaaat 43000 gcatgtgtct gtcactgtgg tgtatttggt gcttgatcaa tgtttacttg 43050 agcaaatgga ggggcagagg taccgatgag tgtgctcagt gaggagggca 43100 ggagtgaagc tgggcgtctt cccgcctctt gtgagtggtg gggcttggtg 43150 agcttgccag ggcctgtctt tcttatcaaa gaaggtgtgt gccccagtgt 43200 tacagcattt cacccaaagc agcctagaaa atgcttgact tttctgtcat 43250 tccgggggagg acactttcct cctccactgt tctgctggcc tggtgtaccc 43300 acggcccctg atagatgata gcacctgcta aagtgcacca tgcccttccg 43350 tctcactgca tcccacagat gaggccaggc tgggatgagg gagaaaggga 43400 gggatatata gttcaggtta ttttggaaaa ctgcctgacc aattttaagt 43450 ctgggccgga cactggggca tctcaccacg ttgaaagggc cgtggcaccc 43500 cgggcggtga aaggggctgg aaccaggtct gcttcttggg cttctcctcc 43550 agggtgccat tgctcatggg ccttggctgc agaggtgctc attcgtggtt 43600 ccaaaattcc aattcctggg agaggaaaaaa tgcttagttc agtctcagtt 43650 aggcctctgc ttagatcaaa cagccaaggc cagtaggccc agtcctatgg 43700 tagagacatg gcctcaaaga gccctctgct gcagttgttg gggagtgtac 43750 caagagaagg gagcattgtc ctgggctggg cagccctggg ggtctagtgc 43800 atagatgtag aaaggctctg ttggtatacc tccctttgct tgttggaaag 43850 tgctcaacgg ggctgaattg tgtttgacag tgtaagtctg ggctggggtg 43900 agggttgtta caagatgtc aagatgatta aatgaaatgc catttgaaac 43950 acttatccat gccttgtgta tggtatcccc accagtgaat attcacagta 44000 tattataata attccaaacaa cttcataatt ttcatatgca atttctaaac 44050 tttgaacttt tttttttttt tttttttttt tgagacagtg tctcgctctg 44100 ttgcccaggc tggagtgcag tggcgcaatc ttggctcact gcaacctcca 44150 cctcccggct tcaagtgatt ctcctgcctc agcctcctga gtagctagga 44200 atccaggcgc ccgccaccac acccagctaa tttttgtatt tttagtagag 44250 acgggctttc gccatgttgg ccaggctggt ctcaaactcc tgacctgagg 44300 tgatccaccg ccttggcctt ccaaagtgct aggattacat acgtgagcca 44350 ctgtgcccgg caattttttg tgtttttagt agagatgggg tttcaccatg 44400 ttggccaggc tggtctcgaa ctcctgacct caagtgatct gcccgcctca 44450 gcctccctaa tgctgggatt acaggtgtga gccaccacgc ccagcctaaa 44500 ctttgaattt ctttgaaccc atgacttaca cagaattagc tgaacgcaga 44550 attccaaatc aactcagcct gtgggacagc caaaaaacac agtgtgcctt 44600 tgggctcctt cactcaccac gcggggttag aaaactttgt cagaggcttt 44650 aaaaaaggag ctcttgtgtg taaaatgttt ccttgattct ctttctggtg 44700 cctctctttc tctaagtggt ttgcttcccc aagttccccca cctgagtctg 44750 ggtggctgtg gcacatctgt gcattctgta cgcacacagg cagccttttg 44800 gagtgccagt ttccaggtct tggttttat tatttattta tttattttt 44850 tgagatgggg gtctcactct gccgcccagg ctggagtgca gtggtgccgt 44900 catggctcac tgcaacctca acctccctgg gatcagttga gcctcctacc 44950 tcagcctcca gagtactagg gaccaccatg cctggcaaat ttttgtaatt 45000 ttttgtagag gcagagtctc accatgttgc tcaggctggt ctcgagctcc 45050 tagactcaag tgatctgccc accttggcct cccaagtgtt aggattacaa 45100 gtgtgagcca ccatgcccag cccaggtcat cttttgaggg catggagaga 45150 agactttgag catcccactt ttgagattgt gtaccagtcg caagccccta 45200 tgacacactt tttccccaaa gtagagggct ctgactatgt tgatcccaag 45250 agagatggga aagagcattg aatgaggatt ccaaagtatt gggccttagt 45300 tcgtttcctc atgttggtgt tgtgaagatt ctggttagga taacagcatg 45350 tgtgcaggag gctttgtgaa ctgctgagag tgaggcgtgg caatgtcagt 45400 gctaggtttg tccttactaa cctggggcca tgggaattga taagaccaga 45450 ttcccaactc taccccacaa tgtgatccct gtggtgaccc ctcacagggc 45500 tctttggtcg agcttcccag aagggatcac catctgccat tgtatgttga 45550 accccattca ttcattcatt cattcagcca accagcaact atttgttgag 45600 ctcttattgt gtgagaagca gtcttcaagg aactgggtga ataaaaaaaa 45650 caaaacatcc taaccttcat tgagcttaca ttcttactga aagaaaacaa 45700 ataaaacata catgtaatcc tagcactttg ggaggccaag gcaggcggat 45750 cacttgaggt caggaatttg aaaccagcct ggccaacgtg aaacccatct 45800 ctactgaaaa ttaaaaaaaa aaaaaaaaaaa aagccgggca tggtggcaca 45850 tgcctgtaat cccagctact cgcgaggcta aggcaggaga atcgcttgaa 45900 tcctggaggc agaggttgca gtgagccaag atcataccat tatactccag 45950 cctcagtgat gaagcaagac tccatctcaa aaataaaaaa taaaaataaa 46000 aatatgcatt ccctttgcac cagcacactt ggtgcctggg gacctcgtgg 46050 ttggcaccct gaagcaggtg tccctcttct gtcttgcaca ccttgcttct 46100 gtcctggtgt gtatggcatg gccttctgcc ctccatggtg agcactgtga 46150 gggcagaggt tgagttgggt ttgctgtatt tctcaggtgc ctaggtttgt 46200 gcttgacagg tagatggaag gcacacaatg tggtcatcaa acctcagtca 46250 accatataag gaaggtagaa gtgaaaagtc ccataggtac ccaactaatg 46300 tcaccagttt cctggatacc tttcctggag tttatttata gtgtgtataa 46350 ataaatgatg tatgtgttta aatgcctttt tcacctttcc ttttagagct 46400 gcctcttttt aacagttcca ttccattgta tggatgtact atgatttatt 46450 gaaccagttc cctactgatt attctgtttt ttgcagtctt ttgttatgat 46500 gaacattcca cagtgacaat gttgttcata gtcattcaca cacatgcaag 46550 tccttctgca ggatatattt ctagagggga attgctgact cagaggtttt 46600 ggtactctgt gttgattgta gagtgacggc agaaaagtga ggcccaagag 46650 tttcctagtg accatgtgta gtggacaagt caccagtccc tgtgagtgtt 46700 tggcccaaag gctttaaggc atttgatatc actgtttttg tttctgcacc 46750 aggcgggaga cactatattc aatcgtgcta agctcctcaa tgttggcttt 46800 caagaagcct tgaaggacta tgactacacc tgctttgtgt ttagtgacgt 46850 ggacctcatt ccaatgaatg accataatgc gtacaggtgt ttttcacagc 46900 cacggcacat ttccgttgca atggataagt ttggattcag gtaagagata 46950 ctcagtcaga atctgtggta aacatgtctc tctcatgtgt tgactaggaa 47000 atgcagtcct ggcagctcaa gagtgcctct ttaagctctg gagcagaatg 47050 cctcctctga gaaatgggtg ctttgtatta gttgagatgg aaagaagaga 47100 ccagaaatgc ctgtagtctc tgcacatcca gacaaaaaca aattttcccc 47150 cctttttttt ttttgtttgt tttttgagac agggtctggc tctgtcaccc 47200 aggctggagt gcagtgccgt gatcttggct caccgcaacc tctgcctccc 47250 gggttcatgc catcctgtca cctcagcctc ctgagtagct gggactacaa 47300 acacttgcca ccatgcgcag ctaatttttg tatattttgt agagatgggg 47350 ttttgctgta ttgcccagtc tggtctcgaa ctcctgagct caagcaatcc 47400 atctgccttg gcctctcgaa gtgctggatt ataggcatgt ggcaccatgc 47450 ctggcctaag aacagttttt agcatttggg aggggctctc atctttaagc 47500 tccaaatgat actgtatttt cttgcttttt tctttctctt gccccacaag 47550 ttttggaaag taaattggaa tagttttccc ccactgaatt atttagcttg 47600 tatacctcag cagatgttcc ttggcctgtt ttgttttgtt tttgagacag 47650 ggtcttgctc tgtcacccag gctggagtgc agtgacacaa tcatggctca 47700 ctgcagcctt gactgcctgg gctcaatcca tcctgcagcc tcagcctcct 47750 gagtagttgg gactacaggc atgagccagc atgtccagct aattttttat 47800 ttttagtgga gatgaggtct ggctatgttg cccaagctgg gcttgaactc 47850 ttgggctcaa gtgatcctct cacctcagcc ttccaaagca ttgggattac 47900 aggtgtgaac cactgctccc gcccttggcc ctataagaag gaatgtgatt 47950 ctgttttcca gcagggcaca aacttctgct taaatacaaa gcccaaattt 48000 ttccaccaaa atgcccctag tgaagtggcc agcccagatg cccgactagc 48050 gtattatcca aagcatattg tcattggtgg aaaatggcct tatagtccat 48100 tgttttgtct taaaagtaaa tatataaata aacttgtata ttgtttccta 48150 attccgtgtt tatattaaca taaaagtgtt ttaaattacc tgtcagtggc 48200 caggtgcagt ggctcgtgcc tgtaatcgca gcactttggg aggccgaggc 48250 gggcagatca cctgaggtca ggagttcgag accagcctga ccagcatggt 48300 gaaaccctgt ctctactaaa aatacaaaaa ttagccaggt gtggtggcag 48350 gtgcctgtaa tcccagctac tcgggaagct gaggcaggag aattgcttga 48400 acccgggagg cagaggttgc agtgagttga gatcgcgcca ttgaacttca 48450 acttgggcaa cagagcaaga ctctgtctca gagaaagaaa aaaaaaaacc 48500 tatcagttga ataacaaaac cctttccttc cttgctttaa gtgaatctga 48550 agatccagga gctgtgctgc aggtaccctc tatgttgggt acccctggtt 48600 taggctgact agtacagtgt ggttggctca tgtagacagc agacccttta 48650 ttttagatac aacttttttt ctttttcttt tatttttttt gagacagagt 48700 cttgcttgtc acccagcctg gagtgcagtg gcgtgatcat ggctcactat 48750 agccttaaac tccctggctc aagtgatcct ctcacctcgg ctttcctagt 48800 agctgggacc acaggtgtgg gccagcaccc ctggctgatt taaaaaaaaa 48850 aaaatttttt tttttagaga tgtctcacta tgttacccag gctggtcttg 48900 aactcctggg ggctcaagca atcctcctgc tttgacctcc caaagtgctg 48950 ggatgacagg catgaactac tgcacctgct gagatgcaac agctttctgt 49000 cagactcatt ttattctcat catttcttcc tgtcctccct tgctgggagc 49050 atgagagctg tgatgggaat ataggaatgt atgaagtcct tctcccagat 49100 caaaaatcct aacttcttgt cttaaaggga ggaaaatttg aatgtaacct 49150 tacttttaga ctcttcagaa atccttctat acccttccgt ccccgctttc 49200 acccttcctc cctctccgtg tgtgtatctt cttctcttga aacacacagg 49250 tttataccct gacccctctt gattcatccc ttgaagcaca gtggtgaaca 49300 aggaaggggc ccgtgatgcc ctaattcttt gccacagcac catgtttgtt 49350 tcacaaggag cctggcaggt ttgggcttgg ggcagatagg ggagagaaag 49400 cagcagagac agcaaaacca aatcatgtca gcttggcatg tacttccctc 49450 tgaaatagct aagaatccat ttctgtaaaa gcactgatta tcagaaaacc 49500 ttatggcct ggccaccttt ggttcaaacc ctcacattaa taatgtggac 49550 agtagtatga ggtgtgccaa aggtggatga ctcagcacct aagtgatgac 49600 acctaattac gaataggttc attaaagcag accccctggg gacctttgct 49650 tgaggatcct tacagtcaga attcctgaat atatttgaaa ataataattg 49700 catctttat ttcatatgtt ctgtatggtt tggctgactt ccccctcaaa 49750 gtctgagtta gagttttcct taatttatgt gatgggtttg gtctttttgg 49800 attccagaaa gagctgggtg tggtttggag ctgcactcag agtcacacaa 49850 aaccacagcc tttagagaac ccacaggaag gctttggggc acgtcctgat 49900 tcttgacatt tctcatcagt gctgactttg tatcccttag gagttcacaa 49950 ttcataacca ctgaaatatt aaaatacaaa aagttttgga aggatgagag 50000 cccagatgct ctactacttg aaaatatgtt aaaacataag ttcatcatta 50050 tacattttgc taaatcagga taaagtctga agtttcaaag aagttttatt 50100 ttagcaaatt ttcagaaaca ctgcctcaac tgttagggcc agtgttctag 50150 tcagtatgcc tttggaagca tgaaagctgg attggtcgat aggatgggtg 50200 tggaaggggg gctgtgactg ggtgggtaca gagaggctct gaaacaatct 50250 cagattccag gagttcctgg ataaggactt catgtgcggg aacagagcac 50300 aggagaagca gattcctgag ccactcagga agaactgggc ctaggcctgc 50350 tcttgtcact gactggcttt ctacataacc acagaaacag cactgtgttg 50400 tagaaagagg aagatcatac tttttgatat ctgtgtctaa tttaaggtca 50450 tctgagccct gatagaaaag caaaacagac aaaacccttg taactgctcc 50500 ctcccacccc acccaccatc aaaaaagctt tagagaggct ggacatggtg 50550 gctcttgcct gtgatcccag cactttggga ggctaaggtg ggtggatcac 50600 ctgaggtcag gagttcgaga ccagcctgac caatatggtg aaaccccatc 50650 tgtactaaaa atacaaaaat tagccaggtg tggtggcaca cgcctgtagt 50700 cccagctact tgggaggctg agacaggaga attacttgaa aacctgggag 50750 gcggaggttg cagtgagccg agatcacgcc attgtactcc agcctgggct 50800 acagagcgag actccttcaa aaaaaaaaaaa aaaaaaagat ccggtttggt 50850 gtcttacaac tgtaatccca gcactttggg aggccgaggc cggtggatca 50900 cgaggttaag agatcaagac catcctgacc aacatggtga aaccctgtct 50950 ctactaaaaa ttagctgggc gtggtggcag gcgcctgtag tcccagctcc 51000 tcaggaggct gaggcagaag aatcgcttga acccgggagg cggaagttgc 51050 agtgagccta gatcgcgccc ctgcactcca gcctggcaac agagcaagac 51100 tacgtctcaa aaaaaaaata aataaaaact ctagagaagc aaaaagaata 51150 actttaaaag tgtttatgtt ctcagcaagc tttatttgg ggatgtcaga 51200 acttaactaa ccactgctcc ttctgtgtgt atgtttttcc tccagcctac 51250 cttatgttca gtattttgga ggtgtctctg ctctaagtaa acaacagttt 51300 ctaaccatca atggatttcc taataattat tggggctggg gaggagaaga 51350 tgatgacatt tttaacaggt aatggtcata acttagatat ctttctcctc 51400 tgtcaacctt cacttccagt tttttaacca atgcttggtt gttcccccaag 51450 gactgaccct cagatgggat gcacccctag tcagcccaca ttcttaggtg 51500 tggcttccta caggtcctgc aggtgctaaa agggatctgt aggaaaaatga 51550 gtttctgaga tttttgtatt ggcctggaaaa aatgtcaaat gggaaccaag 51600 tgacggggca agtttacttt gacttgctgc atgccgtttt gtactcaagg 51650 agtaaaccaa tgtcctttgt aaaaatccct cctttcatta tggtcccctt 51700 tcactgtgaa acaagtttcc ttgagcagaa tcctaactgt cttcacagaa 51750 gctttgtgtt atatttttat tttggagtat tttcacatat acaaaagaga 51800 tactgtagta taataaacct ttgaggacct atccagcccc agcaaccatt 51850 atggcctggt cagttctgtc ccatccacat cctggggctc tttttaagct 51900 ggtaaatcat tatgatgtgg gttgtcattt acagtggtaa aaaacatcta 51950 tcagtagcat ttgaaagaac attctgctca gtcctctggc tgtagaggct 52000 tcaaccccac cagccaccga tgagcacctt ctccctccag gagccagtct 52050 gagctcatta ctgagtttaa tatcagaata caccctggtg cagcctttct 52100 aaattgcagt accagttaac agaaggtgtc tgtcagagca acacccaagt 52150 cattcaagtt accattgtgt gcaaacttaa cagagaccca cgtcttcaat 52200 ataagccttg aaggaaactc cagttttagt atgtagatgg ggtatcaagt 52250 gtgtgcacat tgaacatctg ctgcatacag agcactgtgc caggcaggcc 52300 caggacactg aaaacctgga catagggtcc agacagaagc aagcctgctt 52350 ccacagaggc actcctgggc agacactctg gactgatatg acagtgtgca 52400 gggccgacag gataccacag gtctgaatgg tcagaacagc tggggagggga 52450 gggagcatcc gcaggcatct agtcccatgc taacgcagtg gcactagaag 52500 gatgggtggt gtgtggagca actttcttga aagataaagg acctaacact 52550 ttctatgcac cacttactgt gtgccaggca aggccaggaa tgtttaagtg 52600 gtctgggatc agccagttct gcctcttaac taactttgct gtcctgctct 52650 ccaggctttc attttggtcc tcattccttt tccttggacc aacacagaat 52700 cctccaccct gttctggctg cctctagtct tgttctcagc cctccatttg 52750 tttttttctg ccttttccca catgttctga agccctccat tcgtatacta 52800 ctttccagag acttccccat ggctaaaagc attttggaaa tactgtatat 52850 taggcccctt tcagatactg gcaaccgttt gtgggatgct ctgagaaggc 52900 ctctgtgact tagcctggcc cttttcagcc catcacctgc cacgtcctac 52950 cccagaccct tgtcaccagt ccccaggagc ttacgttgct ccctgagggc 53000 actaggcttg ctctcacttc catgcctttg cctgtgccat cctggctgcc 53050 caaaatgcta tggcagatac ctgttcatcc tcaactgggc tctgcctagg 53100 cttgctccag cagaggttac aaactctatg cttcttcctc tgtgtctcca 53150 acctcatctt cctcttctca cctccatcct ggccctaaag gccctatgtt 53200 tgaagcattc acactgtata ttctgtgggg cacacggccc cagtgtctgg 53250 cacatggtag tcaacaccac aaaccgcaga accagttgta aaaggacatg 53300 gagtcggaat gtgagtttta accagggtca tgctgggctg ggttctggca 53350 tgatgctggg ttgtgggctg agtgagaaca gcaagggtga tggtggatgg 53400 agcaacagtc ttgcagccgg ggctctcagg ccaagtgtat ggcagctctg 53450 tgataatgac tttcccttta ctctttgcag attagttttt agaggcatgt 53500 ctatatctcg cccaaatgct gtggtcggga ggtgtcgcat gatccgccac 53550 tcaagagaca agaaaaatga acccaatcct cagaggtgca ttctttgttt 53600 attcatactc cttccccctt taggatgagg taggctgcag gtccgaggct 53650 ctgggcctag agggaaattg aggtggtcag gttacagtgg agagggagga 53700 ggaagtacgt gtgatgattt cttcttaaga tttttgtttt aagacaatct 53750 ccttgtgctc ttttccttgt aggtttgacc gaattgcaca cacaaaggag 53800 acaatgctct ctgatggttt gaactcactc acctaccagg tgctggatgt 53850 acagagatac ccattgtata cccaaatcac agtggacatc gggacaccga 53900 gctagcgttt tggtacacgg ataagagacc tgaaattagc cagggacctc 53950 tgctgtgtgt ctctgccaat ctgctgggct ggtccctctc atttttaacca 54000 gtctgagtga caggtcccct tcgctcatca ttcagatggc tttccagatg 54050 accaggacga gtgggatatt ttgcccccaa cttggctcgg catgtgaatt 54100 cttagctctg caaggtgttt atgcctttgc gggtttcttg atgtgttcgc 54150 agtgtcaccc cagagtcaga actgtacaca tcccaaaatt tggtggccgt 54200 ggaacacatt cccggtgata gaattgctaa attgtcgtga aataggttag 54250 aatttttctt taaattatgg ttttcttatt cgtgaaaatt cggagagtgc 54300 tgctaaaatt ggattggtgt gatctttttg gtagttgtaa tttaacagaa 54350 aaacacaaaaa tttcaaccat tcttaatgtt acgtcctccc cccaccccct 54400 tctttcagtg gtatgcaacc actgcaatca ctgtgcatat gtcttttctt 54450 agcaaaagga ttttaaaact tgagccctgg accttttgtc ctatgtgtgt 54500 ggattccagg gcaactctag catcagagca aaagccttgg gtttctcgca 54550 ttcagtggcc tatctccaga ttgtctgatt tctgaatgta aagttgttgt 54600 gttttttttt aaatagtagt ttgtagtatt ttaaagaaag aacagatcga 54650 gttctaatta tgatctagct tgattttgtg ttgatccaaa tttgcatagc 54700 tgtttaatgt taagtcatga caatttattt ttcttggcat gctatgtaaa 54750 cttgaatttc ctatgtattt ttaattgtggt gttttaaata tggggaggggg 54800 tattgagcat tttttaggga gaaaaataaa tatatgctgt agtggccaca 54850 aataggccta tgatttagct ggcaggccag gttttctcaa gagcaaaatc 54900 accctctggc cccttggcag gtaaggcctc ccggtcagca ttatcctgcc 54950 agacctcggg gaggatacct gggagacaga agcctctgca cctactgtgc 55000 agaactctcc acttccccaa ccctccccag gtgggcaggg cggagggagc 55050 ctcagcctcc ttagactgac ccctcaggcc cctaggctgg ggggttgtaa 55100 ataacagcag tcaggttgtt taccagccct ttgcacctcc ccaggcagag 55150 ggagcctctg ttctggtggg ggccacctcc ctcagaggct ctgctagcca 55200 cactccgtgg cccacccttt gttaccagtt cttcctcctt cctcttttcc 55250 cctgcctttc tcattccttc cttcgtctcc ctttttgttc ctttgcctct 55300 tgcctgtccc ctaaaacttg actgtggcac tcagggtcaa acagactatc 55350 cattccccag catgaatgtg ccttttaatt agtgatctag aaagaagttc 55400 agccgaaccc acaccccaac tccctcccaa gaacttcggt gcctaaagcc 55450 tcctgttcca cctcaggttt tcacaggtgc tcccaccccca gttgaggctc 55500 ccacccacag ggctgtctgt cacaaaccca cctctgttgg gagctattga 55550 gccacctggg atgagatgac acaaggcact cctaccactg agcgcctttg 55600 ccaggtccag cctgggctca ggttccaaga ctcagctgcc taatcccagg 55650 gttgagcctt gtgctcgtgg cggaccccaa accactgccc tcctgggtac 55700 cagccctcag tgtggaggct gagctggtgc ctggccccag tcttatctgt 55750 gcctttactg ctttgcgcat ctcagatgct aacttggttc tttttccaga 55800 agcctttgta ttggttaaaa attattttcc attgcagaag cagctggact 55850 atgcaaaaag tatttctctg tcagttcccc actctatacc aaggatatta 55900 ttaaaactag aaatgactgc attgagaggg agttgtggga aataagaaga 55950 atgaaagcct ctctttctgt ccgcagatcc tgacttttcc aaagtgcctt 56000 aaaagaaatc agacaaatgc cctgagtggt aacttctgtg ttattttact 56050 cttaaaacca aactctacct tttcttgttg ttttttttt tttttttttt 56100 ttttttttgg ttaccttctc attcatgtca agtatgtggt tcattcttag 56150 aaccaaggga aatactgctc cccccatttg ctgacgtagt gctctcatgg 56200 gctcacctgg gcccaaggca cagccagggc acagttaggc ctggatgttt 56250 gcctggtccg tgagatgccg cgggtcctgt ttccttactg gggatttcag 56300 ggctgggggt tcagggagca tttccttttc ctgggagtta tgaccgcgaa 56350 gttgtcatgt gccgtgccct tttctgtttc tgtgtatcct attgctggtg 56400 actctgtgtg aactggcctt tgggaaagat cagagagggc agaggtggca 56450 caggacagta aaggagatgc tgtgctggcc ttcagcctgg acagggtctc 56500 tgctgactgc caggggcggg ggctctgcat agccaggatg acggctttca 56550 tgtcccagag acctgttgtg ctgtgtattt tgatttcctg tgtatgcaaa 56600 tgtgtgtatt taccattgtg tagggggctg tgtctgatct tggtgttcaa 56650 aacagaactg tatttttgcc tttaaaatta aataatataa cgtgaataaa 56700 tgaccctatc tttgtaac 56718 <210> 2 <211> 56718 <212> DNA <213> Homo sapiens <220> <223> variant B4GALT1 genomic sequence <400> 2 gcgcctcggg cggcttctcg ccgctcccag gtctggctgg ctggagggagt 50 ctcagctctc agccgctcgc ccgccccccgc tccgggccct cccctagtcg 100 ccgctgtggg gcagcgcctg gcgggcggcc cgcgggcggg tcgcctcccc 150 tcctgtagcc cacacccttc ttaaagcggc ggcgggaaga tgaggcttcg 200 ggagccgctc ctgagcggca gcgccgcgat gccaggcgcg tccctacagc 250 gggcctgccg cctgctcgtg gccgtctgcg ctctgcacct tggcgtcacc 300 ctcgtttact acctggctgg ccgcgacctg agccgcctgc cccaactggt 350 cggagtctcc acaccgctgc agggcggctc gaacagtgcc gccgccatcg 400 ggcagtcctc cggggagctc cggaccggag gggcccggcc gccgcctcct 450 ctaggcgcct cctcccagcc gcgcccgggt ggcgactcca gcccagtcgt 500 ggattctggc cctggccccg ctagcaactt gacctcggtc ccagtgcccc 550 acaccaccgc actgtcgctg cccgcctgcc ctgaggagtc cccgctgctt 600 ggtaaggact cgggtcggcg ccagtcggag gattgggacc cccccggatt 650 tccccgacag ggtcccccag acattccctc aggctggctc ttctacgaca 700 gccagcctcc ctcttctgga tcagagtttt aaatcccaga cagaggcttg 750 ggactggatg ggagagaagg tttgcgaggt gggtccctgg ggagtcctgt 800 tggaggcgtg gggccgggac cgcacaggga agtcccgagg cccctctagc 850 cccagaacca gagaaggcct tggagacttc cctgctgtgg cccgaggctc 900 aggaagtttt ggagtttggg tctgcttagg gcttcgagca gccttgcact 950 gagaactctg gtagggacct cgagtaatcc actccctttt ggggactgac 1000 gtgaggctcc cggtggggaa ggagactgac ctctcggttc acgtgtcttg 1050 ccatagagcc actctcctga gtgggttttt ctcctgatcg tttgggccaa 1100 gtgacttctc tctgaacctc atatttctct tctggggataa taaatggtca 1150 ccctttcaag gggttgtttt ggaagatatt gtgaacaatg gtaaataagg 1200 gcttaattaa tgagggtaag ccctcagtaa attgtcactg tgtgttcatt 1250 tcttcctctg tgtggatcgt gaccgagagc ccttccccct agcctcctcc 1300 tggtatgggt acccaaaacc taggtgagca gggatctctc ccaggggcag 1350 agagcttgtg tactctgggt gttagagggc taaaatataa ccagtcaaca 1400 ccacgttgcc catttctggt acttccggta gcagcctgag tctcaattat 1450 cttgcccaga tgatctgaac tctgacctct agcctgtttc agcataggca 1500 gagagcttga gtaggtgagt ttgcattcct catagcagct ggctgagcct 1550 agtctggact tctctttgac ctgtaaccta caggcccaca ggcccaaggc 1600 aaccacaggt tgcttccagg gttaccacac aggtggtttc tcatttctaa 1650 tgctaggttt tagataattg ttgtaagtga ggggccctgg caggcaggat 1700 gacatcctgc caataggagt tttctgtcac tttcccacag agccctggct 1750 actacatact cttgctcaat ttcgccagta attgcgtcaa tgtgttcata 1800 tcaagtttgg gaagaacatc ttggaattgg tcagacgtga actgtggtaa 1850 taatgggggc ttgttttttt aagcagataa ttaaattcct ttgcatttga 1900 tgattattct gggaagcaga ctagtcccat aaaatgaaat ggactctgcc 1950 ttgctgctaa gtgtctgact tgagacatgc tatcgagttt ctcaaaatct 2000 cttccttgtg taaaatgtgg ttgtcgatga ttaccttaca ggggtttttt 2050 taagactaaa tgagatcgtg tacattaaat acaggcactc aggctgggca 2100 tggtggctca cgcctgtaat cctagcactt tgggaggctg aggggagtgg 2150 atcacttgag gttaggagtt tgagaccagc ctggccaata tggtgaaaca 2200 ccatcccatc tctacaaaaa tacaaaaaag ttagccaggg gtggtggcat 2250 cgcagctact caggaggccg aggcaggaga attgcttgaa cctgggaggc 2300 agaggttgca gtgagtcaag attgtgccag tacactccag cctgggcgac 2350 gaagcaagac tgtctaaaaa aaaaaaaaaaa aaaaaaaata cgggcactca 2400 atacacgta taataataat atagtaataa tatttgctta ggatctttaa 2450 aaagtttcat tttttcagac tcccacagaa atggctctgc acagcagagt 2500 gaagggggag agagactgag tctccaggcc agaaaaaggc caggtttttt 2550 gcttttgttt ttagttgttg cctggatatt gcacagaaag aaaaaataat 2600 tagcaagtta aaaaaaagta ccgcaaagtt gattacattg gtatttgagt 2650 atcacatctt ctctcagaag cgtaagagac aaggtcgtga ccatacctct 2700 gcttagtttt gttttgtaat ggtgttgcta gtgatcggct tgtcaccagt 2750 tactggtgtt tctaaatgga ctataattgg ctacttgaaa ggacttcctg 2800 agaaagaaca ttttggagga cgaggagaga gtgccttctc tattttggct 2850 gctttcatgt gacatgcaag agaccatgac gtttaggctg ctgctgaggc 2900 agccccagaa atgggggccg agaggtcttt tcttcatttt aatagggtct 2950 gtaggtttgg gtggttaggt acagttctca gaatggaggt tcctggctat 3000 gaggccttga gaaagctgaa agtctccttg ggagtgtgtg ggtgggggga 3050 gtcgagccca tctgttcatg ggcaggtgtc agccaaagcc cttgcgggtg 3100 gttttgaggt tggtggggaga aagcatccgt ggggtttaga gttgtggcct 3150 tttcactact tgcagttcct ttccccgact tggctttact ttctggtgtc 3200 caggggtctg ggccagatgc tgagattcct ctcagctgac aggtgtgggt 3250 tatgggcaaa cccttccctg gaggacataa ggcaccggat tggactgctg 3300 atgggttgct gttggagttg tcagggcctt ggaaatagtct tcagatagac 3350 ttgggttagt gtgacctggg gcaggctgca ggtttggagc catagtaccc 3400 cccgccccca caccgggcac cctgctctgg gctaatgtga ggcttgcagg 3450 agtgagtgat gcagtgggaa ggggggcctt tcctgaggat tctacagctt 3500 tctccaggga atcctcccag gtagtttagg cctgcaggtg ctatgctatc 3550 cttctttcct aaccctgtct caggtcctca gcggggccat gcggcatcca 3600 cttataaccc tgcagcgagg ccctcttttc tggccacctg ggtgtttgcc 3650 tgctgagatg ggaggaacag tggccttggg cttcttcccc cgtcatgttt 3700 atctctgctc agatgggca gcagctcaat gggacttgac cagctgtggc 3750 actgccagtc tgaagatgag tagggtgatg gggggaggtg ggcagtacct 3800 gaagctgaac tggtgagaga ggcaggctgg cctgggggct cagctggggc 3850 ctgggatggt tggtacagtc ccctcagggg ggtaggggag tgagtgttag 3900 actgcttaag cctcagaggc cgctcttgcc cacctatgct ttgaggagat 3950 cctcttcatt tgttcaaagg gaagactctg atctagagat gggcacttgg 4000 accagcaaac agcagctaca ggtagccagg gcacccgagg agcacttgct 4050 catgagccgg tttccctggt ttttatgggg gctgttgctg agcgtctgcc 4100 agggtttgtg tcctagcact tgctggtctt tgctgggctc tcagctctca 4150 ggtgtttctc taccagcacg tttccccctc cctcatatgc acacatgtgg 4200 acacaagcag gctgcccagg acagagtgta ctttgaggct tgggaaagga 4250 ctctctctcg cccttttggg gatgagcctt ggaacctcat caccttccgg 4300 cttggggtgg agcttcatcc tgggggttga agctttaggc tcagataact 4350 agtcttgtaa gccagttttg tcctgttgtt tttttcgtgg aaaataatgt 4400 attgacgtat acacagacat tctttgtcta acagtctgag attgagaaat 4450 accctccatg actatttggt ttgctttcat ggtgaaactt ggtcgctttc 4500 ttagacacag cctatggcaa taagagtgat ccctggctgc tgtaattcat 4550 tccagacttt gagcaaacac aaggcaccgc ctccacctgc agtggagcct 4600 ctgatgaacc aaatggaaac tccttgggga atggggagta agagccaaat 4650 gtgggattgg acttaaactg cagcttctta gaactgtagc attccacgat 4700 gggattgtct agtgctcttc ctggaggtta ctattcaata gttggctagt 4750 gcacaggttc aggggtgacc tgatatgccc tagcgtttca gaagatccct 4800 gcaaggtgtg tcttttggtc catctgaagg gtcttgtatg gtgatcttgt 4850 atggatatcc gtgacggcta aggcatctga taacttcatt ccttcagttc 4900 cagcagtgtt cctgtattat gctgggcact agagctacaa agaagaaaac 4950 aaagtgcctc ctcttcagga actcttaatt taggcagggg aggcataatt 5000 gaacagtgct gaggtcatct aggggaacca aagtgtgtat ttatcccctt 5050 ccctatcact cccctccctc cttcatttct tcctttcttc tttcagaaac 5100 tccaagttca tatcaaaatt ctccagccct ggttttatattt ggttgtgtga 5150 aaattttcct ctaatttctg aagctatgca ttagttctgc tgagtaatct 5200 ttaacttgct gctttataat gattataatg agatatcact gggtattatg 5250 gtctttgggt agcagcaggg tagggatttc caggctggga ctaagctaat 5300 ttatgggttg ggaattatgg ggcagttaat agcaaggcag tccaagcttt 5350 ccacagattc caccctaggg accatccaga cttaaggaac agggccggca 5400 ggctcatccc ctttgcactc agctgggcta tgggtgtgtg tttgtgaaag 5450 aggtttattc agtagtcata cctgctgatt tccctgctat ctgtttaccc 5500 agtgcctcct gtaccttgtt tcttactctt tgttctctgc tcttactatg 5550 aagaagcaga gactggaatt ctgcttgaac ccacatctac ctggaaattc 5600 cagtttttct tgtccagtgg agcagcaatc cagttgtttt aggacaaatg 5650 gtctgccctt gaagcttaaa tcctttgagg gcctggcatg gtgacagttt 5700 tacatttggc tttggtatag actggtgtgg tccctgggca gtgaggtcac 5750 tgtaaggcca gccagccaga ccctggctcc taggggaatt aacaaggcat 5800 gggattagac tcacagggtc cctcctgtcc ctaaacttgg taggggttcc 5850 tgggagccag actgcgatta agattgtaga gacctgagac ctgagttgta 5900 ggggcctctg tgttgatctg ggccattgcc gggtgagctg aggcggtcac 5950 tagctcaagg agtgatctca ggatattgtt ctgtaagtca gagacctcca 6000 ggttggagag tggggcttgg gggtggggga cagggtttag tggggagctg 6050 gttctgggtg aatgtggcct aaagggattt gtccttagaa gacagagggg 6100 tgagtcacac actcagtgct tcaggttcca ctttgcggct tggcctcagc 6150 ccgccccttc cctgcacaaa tgaaggccag gggctatata attggctgtt 6200 gctgaattct ttggcagtga ttttaaagtc tggtctgggt gtgttatgta 6250 gctgcttctc tatccactcc ccacacccgc tgcttctcca gagcccctca 6300 caaagcccag gcagagagag agagagagag agagagaatg acttgcctca 6350 cagagatgtt ggggatagg ataggggtat gggtctttgc ttttgccttt 6400 tgagggggga taatctcttc cttcatttta aaagtaaaaa gtaatgcagg 6450 ctcattgaaa ataatttgaa aagttgaaag agatataaaa gcacacccaa 6500 attcctatca cccaaaagaa acataccggc atatttccta ctagtctttt 6550 tcatgtttaa gaatatagct gatatatttt tttttctttt tctttttgag 6600 acagggtttt tgctctgtca cccaggctgg agtgcagtga tcacggctca 6650 ctgcagcctc gacctctcgg gctaagcgat tctcccactt cagtctcccg 6700 agttgctggg accacaggtg cacaccgcca tgcctgacta atttttgtat 6750 tttttgtaga gatggggttt tgccatgttg cctaggctgg tctcgaactc 6800 cagagctcaa gtgattcacc tgccttggcc tcccaaagcg ctgggattat 6850 aggtgtcagt caccacaccc agtgttatag ctgttgtctt tatagatgaa 6900 cagatagatt gacatagatt catgtagata gcctggtgtt cagcattttt 6950 catttaagat tctgtcacag acttgaccct atacctttaa aaatcacaaa 7000 ggcagtatca tagtctgtca gctgaatatg ccataactta aaaaaatcat 7050 tcaactgttg ctgaacacac acatatacat atatagtttt tgttttttct 7100 tagtgatgta gtgatgcttg tgcagaaagc tttatgtact ttttggatgg 7150 tttctgtagg agagctttct aaaaaagggaa aaaaagtgtt gaatgttttt 7200 tgagaagggc tagattttca agccagtctt acaaaagggat agactcattg 7250 gaaattccag atttgcttag tgctggcaga tgagtatcac ttattgctga 7300 acaatgtgtc tagaattctg attaaaaaag aaactaggtc caggaagtgc 7350 ctgggggcag gggcaaaggg ccaggctgca ggataggctc ttaggatctg 7400 gctgagcaga aatctgctgt gaacagaatc ggtgggggtg atgctttctc 7450 agtaacttct ccatttgttt ctttagcagc taagtccctg tgctggactt 7500 ctgtggacta ctgtggctct ggggctgtgg ttgtgggtga acaacagcta 7550 gctaaaccag tgctgttgac atcattgaga tgtgacgcac aggaaggtgg 7600 gagcaagctt gcaaatcaga ttctgaaaca tatagcacag ctctcccacc 7650 tccaggtggt cctgagatct agggaggagc catagtgaga aactttaggt 7700 ttctaggaat tctcttaggg agaagctctc ttagggagag gcagaacctg 7750 gttctcagtt ggggctgatt caggtgggtt agatcaataa agcctcaggc 7800 cagtgtgcca ggctattccc aaggagtata ctttgaagtt actcccttta 7850 gaatgtcctc agtggagata aattctctct gaggagcagt tttgtctgcc 7900 ggggtcattt ggcacaaagc ctggagtgct agggcgaggt tgcactgagg 7950 gaagggggcag gattatgtca gcagtgtgac ggatacagtg tgaggtcagg 8000 ctccttcctg ccccaccacg ggggcctaga ggtcatgggg agggtccctg 8050 gcaggggatt caatcattgc ttggccccat gacagagtat attctaaaaa 8100 tgccttaagt ttttttcttt caaagtttct tcctgttttg cataatggcc 8150 ttttgccttt gacatcctga aaccgcagag ctgtcattgg tgttgcagga 8200 cactgccagc ttgaaaaaaa tcaacaaacaa aaaaagaaac aggaaaggat 8250 gtggagttca gggtgcggcc tagggaagct ggtatttgcg ttatgggatt 8300 gtggggatgt ggtattaagg tgttgggtag cgcctgacat ttagaggagt 8350 actctgggca gagtccctgc ctgcccaaga ataggtagaa ttgagtcttc 8400 acaccaaagt caggagagac cccctccccc caggaagaga atgaacaggg 8450 actcatttcc tcattcagca aacttttat ggtaactaca ctatatgaag 8500 tgtgagagat agacatgaac aagagaggcc cccactcttg ggcagtccct 8550 tagtagtagt agatagactc tggcaatatg gtgtggtcag agagaggaag 8600 cctgggtgct ttgagggtac tgaggaggtg cagggagcca aatgggtggt 8650 ctgggccagg gccagagtca gaatgaagga cctctcttcc agacgttgat 8700 tttagcatct ctgtctctca gtatgtttga acagtctccc ttattggaag 8750 ggcaggagtc tactgctaaa agtaacctgc gatttcctct acttgctgtc 8800 atgtggaaag aatactaaag ctgaaattcc aaaagttgca cacctttacc 8850 agcagggcag gagaggaaag gaaatggagg cagagtgagc tgaagatgat 8900 aaaagaaaga gaaggtggtg cagtttggac tgttatggac agaggaagtc 8950 tgagggtagc tggactgagg gatcaaaggg aggcagttga aagggaagag 9000 agctgcagag agggatttct tggtctgcag agggtaggag caagccttga 9050 aggctgctgg agtgaggatt ccgagccctg gtctttattc tttttctaat 9100 tcatttacatc attttaggca agtcctaact cctttggtct ctgttgtctt 9150 tctgaaattt gagtgggctg ggcctgctgg tctttagcct ctgtctttct 9200 ctacctccta gattccagtt tggcgagtgg gggggaaaac ctggttgtat 9250 atgcaacgtg aaaggcctct ggaattcctt ttgaagctca ctacccatga 9300 ggcttctgct aaggatttca tcatgtctgt ctaagcagac ataaaaattt 9350 tagcaggtgg atgacccgta gaaatggcac aaggaatgtt tctttctgtc 9400 acactgtggt atttgattta agaaagttgt tatcctctct gtgcctcagt 9450 gttctcactt gtaaaatggc aataacagta tccacctcat agatgttatg 9500 aaatacaggt agtagccacg aaagggctta aaacagtgcc taacacagaa 9550 taagttgtga atatatgtta tttattatg gtagtataat gcttatttgt 9600 gaagattttg gcttttgctt tataggacct tttttttttt tagttgaaaa 9650 tacaatgtta ccatgttaaa tgttaaaaaaa aattctactt accattgtaa 9700 cagaacatgc tcccacttct gtaacagagc ttgctattac ttttcaaatg 9750 catacatatt ccaatgcata tattccaatg cagttgtaga gtgaaactgt 9800 ttgcatgcag ccatttttat ccaaacattat cttataaaat gttatgttgt 9850 ttatgattat cctaattatc ttttgttgct gtctagtatc cttatagata 9900 ttccattagc atacactatt ccaggtttca ctatcgtcga taatctagat 9950 atgaacattt ttgtagtgtg tagctctttg cttcagttga attactttcc 10000 tgggataaat tcctggggaa gaatttctag gccagaggat atggtcatct 10050 tgacaatact gattcacatt gctgcattgc tttccaagag gtttggaatc 10100 attcacaggt tctaaattgg aaaatcctgg cttttgaagt atgtggattc 10150 taagggcgat ttggatctag ctggagcctc acactgacac ttccagccag 10200 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtagt tccctatgct 10250 ggacaccgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtagttc 10300 cctatgctgg acaccatgtg gcctttctgg acattagggt tttcctgtga 10350 ttgcctcaga gcagttcctg ttgaattcac tctgtgtcca caaaaggagc 10400 cttactgtgg ctctttcaac acccacctac ctttgccaag ttggtttaca 10450 gaaagtaaga acattctttc cttcttcctt gatatgtggc gctaaaaccta 10500 tagcatgggg caggctctgg ctttaaaaac ctgacttaaa aataatggtg 10550 ttgatcaaaa agtttgtgga tcagtttttg gaaacactgc atgtagccat 10600 ccatagaaac ttatattctg ttgggctagc ctgggcgcct gatcatttaa 10650 ctcatgtgga tgaacttcta tgtaatagcc ctggtgtatg ggatccagaa 10700 acaggggccct aatgaagaaa ggcttttaaa ttatgttgga taaaaataag 10750 ttgttacaat agcccaaagt ctgcaaatat gaattgccag ttctgtcctt 10800 gtagtcatcc accatgtgcc tgcatctttt gtagactctt gtagattcag 10850 aagcccactg aattgcataa atgatggaat gattttagac ttagtgattt 10900 cagtgactaa aagtttacag atcctggccg ggcacagtgg ctcacaccccg 10950 tattcccagc actttgggag gccgaggtgg gtggatcacc tgaggtcagg 11000 agtttgagac cagcctggcc aacatggtga aaccttgtct ctactaaaaa 11050 tacaaaaatt agccgggtgt ggtggcatgc acctgttgtc ccagctactt 11100 gggaggctga ggtggggagaa tggcttgaac ctgggaggcg gaggttgcag 11150 tgagcccaca tcaggccact gcactccagc ctgggtgaca gagtgagact 11200 ctgtctccac ctcccccgcc ccccgaaaaa aaaaaaagtt tacagatcca 11250 gcagatgggg catattcaat ttgtgacagc cactcccttc accttatagc 11300 tatgtcatat gtcttcttct cctttgactg cattctgcag cagtcagttg 11350 tgacttaata tggcactctg ggcccactga attaggtcag agctgctagt 11400 agtatattgt tcctagagac ctagggcaag attttcttac tacataaaat 11450 gagggagata atttcttacc tcaagatgtt ggtaagagga gtgaatgagg 11500 ttagttatat ggtaatatca gtactctgaa tgtcttttga tcaatgccta 11550 actcatcttc ttgggcacaa aaggcataca gtcagcaccc ttaggccaca 11600 tataaaattc ctccaaatgc aggttttcat ctgccttggg gcagagtcaa 11650 gagaaagaag aggaagaggc gtgaggctct gaccacaact tagggacaga 11700 atatagccca aagcgagtac cccaggccac aaggagaagg ccgctatctt 11750 gttgaatcca cagcactgga aacttggagt gtgtgttccc ctgtgtcagt 11800 tacactggaa ttttatggct gctcacattc ttcccttcag gtggacgttg 11850 ttcatcagta tcctgggcaa gaggccatca taaaccacag acagctgagt 11900 gattaggaag aggagctgaa gagggagcat tagatgtttg attgagtctt 11950 aggtgagaaa gtatatcatt aaaacaaaaa gatagatgta ggcgggctca 12000 gtcttgtgtg cctggtgtgt tggtagaaaa actaaagcac aagcctgtag 12050 ataacctgct ttattctacc tcggggctgg tgttggaatc caggatgcca 12100 gaccctaaag tccagctctc tttccaacct actgaataat ccgagagaaa 12150 tcatgttctc tctctgggcc tcagtttgcc catgtataaa atgagatgaa 12200 ggattggctg ggatgctctc cagagtctct tcctgcctgg agttctgacg 12250 tagccatgta ctcctgctca gcatcgctaa atggctttgt ggtaggacca 12300 ttgagtgctg cctccattag ggccagctat gtaatgctgg ggtggctgtc 12350 actgggccct aagagccagg attggtctta ctggagaaat ccacatccac 12400 ctaaacttaa gacccagggg tgtccaatct tttggcttcc ccaggccaca 12450 ctggaagaag aattgtcttg gaccgcatat aaaatacact aattatagcc 12500 gatgaggtta aaaaaaaaaaa actcaatatt ttaagagagt tcatgaattt 12550 gtgttgagct gcattcaaag ccatcctggc cgcatgtggc ccatgggcca 12600 tcggttggac atgcttgctt tagacctccc agcaattcta gtctctaaac 12650 aggaaatcaa aagtcaagat gaatagataa gttggtcagt gtgaaaaagt 12700 aattggtggg agccactgta gatgcagggt tctaggctcc atcaacaacc 12750 acctacatca ctgaacgaaa gataatgctt gttcagcact tattacatgc 12800 caaccatggt aaaaatactt cagatgcatt gttttcatga actctcacag 12850 cagctctttt tcttgcctaa atgccccgtt agaacctcca gtacaatgtt 12900 aaatagatat gctaagagac aacatatgtg tcttgttagg gggaaaatat 12950 ccagtctttg actattaaga atggtgttag cagtgggttt ttcctaggtg 13000 ccctttatca ggttgaggaa gttcctttct attcctggtt tgttgagtat 13050 ttttatcatg aaaaggtgat gggttttgtc aaatgctttt ctgtgtctgt 13100 tgagatgatc atgttttttt gtcatttatt ctattgatat ggtatattat 13150 acattgattt ttcagatatt aatcttgcat acctgggata aatcccactt 13200 ggtcatggtg tataattctt tttattgtt gctggattga gtttgctagt 13250 attttgttga tttgtattca taacagatag tggtctgtag tctttccctc 13300 cctccctccc tccctccctc cctcccttcc ttccttcctc tctctctctc 13350 tctctcccct cccctccctt cttttcccct cctctcccct ccccttccct 13400 ttcttctctt tcatagttgt ttaccactgt cagaaaaggt ctgttcgttt 13450 tctttcgtcg tgagatcttt gtttggtttt ggtatcaggg taatactgcc 13500 tcaaaaaatg agtaggggaag tgttccttcc tcttctgtat tttgagagag 13550 tttgtggtcg gtttttatta attcttcttt aaatatctgg tagcgttcac 13600 cagtaaagcc atctgggcct gatgttttct ttgtggaaaa ctttttgatt 13650 cctaattcag tttctggtta taggtctatt cagaccttct attttttctt 13700 aagtcagttt tgatagtttg tgtcttccaa ggagtttgct tcatctaagt 13750 catctaattt gttggcatac atttcatagt gattccttat gatccttttt 13800 atttccgtta aagttggtgt agggatagtc cctctttcat tactgattat 13850 aataatttga attttctttt tttcttagtc ttgccaaaag cttgtcattt 13900 ttattgatct tttcagagga ccaactttga gttcattatt tgttctcttt 13950 gttcttattt ttctgcttca ttaacttctc taatctttat tctttcattc 14000 tgcttgcttt tggttaagtt tgctttttct ggtgtcttaa ggtagaaggt 14050 taggttactg atttgagatt taaagatcat gctctttaaa cgttttgata 14100 gatactgtca gtttgccctc tggctttttc tcattaacag tgtataggag 14150 tgcttattcc tcacactcat accagccctg ggtgttacta acctttatat 14200 atttgccagt atcatattca gacatagtat cttgttttaa tatgtttctc 14250 tgattactga tgaagttaag caaattttca cgtgtttat ggccatctgt 14300 ctttcttttt tcatcctttc tttcaagatg ggagtctttg ccatgttgcc 14350 caggctggac tcgaactcct gggctcaaat gatcttcctg cctcagcctc 14400 ctgagtagct gggactatag gcgtgagcca ccatggctgg cttgcccatt 14450 tgtatttctt atgtgagtat tttttctttt tttttgaagt ggagtctcac 14500 tccatccccc agagtggagt gcagttgtcc gatcttggct cactgcaacc 14550 accgcctccc aggttcaagt gattctcaca ccttagcctc ccaagtatct 14600 gggactatag gtgtgtgcca ccacacctgg ctaatatttg tatttttagc 14650 agagatgggg tttcaccatg ttggccaggc tggtttcaaa ctggcctcaa 14700 gtgattcacc tgcctcggcc tcccaaagtg ctggggattac aggtgtgagc 14750 cactgtgccc agctgacttt ttttttcttt tttttaaccc ttttttttt 14800 ttaccctttt tttggcccat ttttttttac cctttttctt ttaacccatt 14850 tttctattag ttttaaaaat atgtttgcag gagcttttta tattgtggat 14900 ttttcttgtt tattacatat catttgtaaa tatggtctct ccatctgtca 14950 ctcttcttta tctctggttt ctttagctat gtagaagttg ttatgttatg 15000 ttatgttatg ttatgttatg ttatgttatg ttatgttatg ttatgttatt 15050 ttttggagag ggagtcttgc tctgtcgccc aggctggagt gcagtggtga 15100 aatctcggct cactgcaacc tctgcctcct gggttcaagc gattctcctg 15150 cctcagcttc ccgagaagct gtgattacag gcacccgcca ccacacccag 15200 ctaatttttg tgttttagta gagacggggt ttcactatgt aggtcaagct 15250 gatctcaaac tcctgatctc aaatgatcct cccaaagtgc tggggttaca 15300 ggcgtgagcc actgcactcg gccagaagtt ttgaattttt atgtgtttaa 15350 atctatgttt tcctttatga cttcaggttg ctttcatact taagcaggtc 15400 ttcaccatcc caaaatgata aaatttttct cctgagtttt cttctaagtt 15450 ggttctttag aagccaccaa cttggcttcg acagcaaaag atgaacagaa 15500 tttctgttca actctcatgc tgcaagaagc tttatgtaat actccaggga 15550 ccctttaagg tcccagagtt ttcctccaaa tctatcagtg attctagtgg 15600 ctaagagtag aaatgtgaaa atttagccat gtgtgctgat agagctgtag 15650 taatttgtaa gctctgaagt tctaaggagt caggggagaa gggaaagtaa 15700 catttatga acatctatta gctcaataag aacatgcgat aagtatgtat 15750 atgtattatt tcacttacat ctgaaaggaa ggcataatta tccccactcc 15800 ttagagaagg aaattggagc tggctacatt taaagtagtc ctgacaccag 15850 agagatattg ccaggagtac ttggctggct gagtgcccag atggcccata 15900 ggagtagtgg gccctccaca gtccaaggtc tggttctagg tggagagaga 15950 aggatgtgct cgtagtcagc accgcagctc cagaaaatct gctggggctc 16000 caaaactgat tagaggggca gctgactcag taataaaact cccagggagac 16050 ttacttacat actggaatgc aaagttgcag ctttactggg aagattagaa 16100 ctgttatga gtagcttaga aatctctggc tgaattcact gcaaggggaag 16150 ccgcaggata agctaactgc tggtgagtca gcagtcagag cagggaagtg 16200 aatttaacat tagatgggtc agtctctcgt ggctgatgaa ttcatcccca 16250 caatactgta cacctgcctt agggaccttt gtctggacta ggggttgggg 16300 tccccctcct ttgtacagcc ctggaaggac acatccagct ccatccgcca 16350 tctctccctt acttatttcc ttccttcctt ccttctttcc atccagccat 16400 caagcttcct ttcatggcca ataatcatca ttggggtcta ctcatggact 16450 ctcttgcctc atgtatttgt tttattttgt cctcattccc acttctattt 16500 cccaggtata tcacaggcaa ctattctaac gtatttatag tttgtgtatc 16550 tgtttttgct cttgccaaaa tggaagccac tgctttatac atagatgtat 16600 tcttaacttt aaaaaaaatt tttttagatt aacctacaat aaaattggct 16650 ttttggcata tagtctataa attttaacac atacatattt ttgtgtatct 16700 accaccacaa tcaggataca gaacagttcc atcaccccaa aaaaatccct 16750 cttgtagtca cattctcctc ccacccttaa tcccaggcaa ccactgatct 16800 attcttcatt actattgttt tgtctttttg aggatgtcac ataaatggag 16850 tcacacagta tatatacatt tttttaaaca tatgtaaaatg gcattttata 16900 gctcattttg attatatgtt tttcatccag ttctgttttt tttttttatt 16950 tttaaaaagt ttgacataac ttcagactta cagaaaagtt gttagactaa 17000 tacaaagaat tcctggatat cctttggagt ccctaaatgt taacatttta 17050 ctatatttac tttttccttc tctctctctc tctctctcgc tctgtgtgtg 17100 tgtgtgtgtg tgtgtgtgtg tgtgtatcta cctgtagata gatagatatt 17150 aatataattt tagatagatg tatctagatc tctctctctc atatatatgt 17200 gtgtgtgtat atatctatat ctatatctat atatatctcc ttttaccctt 17250 aaatattcag tgtatatttc ctaacaacaa ggtgatttaa aaatatatat 17300 ataaacatag tataattaac aatcaggaca tcaacattga aacatttctg 17350 ctatgtcatc tacaggcctt aggaagactt tgtcaggtgc cccaataata 17400 gccttgatgg tagaagaaaa ccatgtgttg tattcagttg tcatgtctct 17450 tagtgtcttg taatctgaaa taattcccaa gccctttgga tttcatgaca 17500 gtgacattgt tgaagagtac aggccagtta ttttgtagaa ggtctctcag 17550 tttaggtctg tctgatgttt cctcctgatc agattcaggt tattcacttt 17600 tgacaggaat accactgaaa tgatgctgag ttcttctcag tgtaacgaga 17650 tctagagaca cacactgtca gtttgttcct tattggcagt gtgaaccttg 17700 aggatttcat tgtagtggca tttggcatta ctccattata gttactattt 17750 taccatttta aattaaaact atctggccgg gcgtagtagc tcatgtctgt 17800 aatcccagca ctttaggagg ctgaggcggg caaattgctt gaggtcagaa 17850 gtttgaaacc atcctagcca acataacatg gtgaaacgcc atctctataa 17900 aaaatacaaa aaattagcct ggcgtggtgg cgcatttgta gttccagcta 17950 ctcaggaggc tgaggcacaa ggcttgcttg agcctgggag gcggaggttg 18000 cagtgagctg aaatcacgcc actgcactct agccagggtg acagagtgag 18050 actctgtctc aaaaaaaaaaa agtaaataaa taaaaaaatt ttttaagtat 18100 cttatgggca tatacttgtc ctgttactcc tcaaactttc atccactttt 18150 ttttttttaa attttttttc ttacctttca tcgttttctt gatatccact 18200 gggttttagc atctacaaat gattcttgcc tgaatcagtt attatggtag 18250 ttgatggttt tctaattcca ttattccttc tatgtttgtt aattttggca 18300 ttcttctata aggaagagct tacccttttt ccctattaat taattcatat 18350 attaatgcag acctatgcat tcttacttca ttaaatcata atcctttact 18400 atcattatgt attctgatgt tcagactatc ccagattag ccaataagat 18450 ccccttcagg ggaatggtct ttgggattcc tctttagagg ttcctggttc 18500 ctgttttctt ttgacatatc ctattactct ttgagcattt tttttttttt 18550 ttttactttt aggcacagca agaagttcca tggtcctctt gttctttccc 18600 caactcagcc ctagagtcag tcacttctcc aatgagctct agttcctttt 18650 agtagagaat cataattaga aaacaagaat cagtgccaag tgtgcacctt 18700 tgtttttaag gtccatccac gttgccgtgt atatgtccag catgttgatt 18750 ctaactgctg aataatacct catgattgtc atccatccca gtgtttcttt 18800 ttcccttctg taatgaggga ctcctggact gcctccagca ttaccttcac 18850 aaatattgct gtgaggaaaa tccttaaacg tttcctttat gggcaacgtg 18900 tgagcatgtt tatgttgatt caggggtgcc agacacagct ccagaatggc 18950 tgcctcagtt tacatttcca ccagcagagc atgacaggct ctgtgtctcc 19000 gtgaataatc agcattaacc agcttcctat tttttgccaa actaatagat 19050 gtgctaggat aactctttgt tttaacttgt ttttctctga ttaccaatga 19100 gctggagcat ttcttcatat gcctgatggt ctttgggatt cctcttaggt 19150 aaattgctta ttcattataa tcctttgcct gtttttcact ggagttctta 19200 tatttttctt gaagatatgc aggaattcct tatacatcct agatattaat 19250 cccttcctgg tctcagacat tgcagatatc ttctgaatct gttattatact 19300 tatttattta caattttttt tttaagagtt ggggttttgc tctgtcaccc 19350 agactggagt gcagtggtat gatcatgact cattgtggcc tcgcaatcct 19400 gggcttaagc gatcctccca cctcagcctc ctgagtagtt gggactacag 19450 gtatgcacca ccagacttgg ctaattttat tttattttt agagatggaa 19500 gtcttaatat gttgctcagg ccaatcttga actcctggcc tcaagcaatc 19550 tttccacctc agcctcctgc atctattata tatatgttca ctttgctcat 19600 gctgtatttt gttgcaacat aaaactattt ttcccattgt tttgtgcagt 19650 ctctcaccag cactcttctt tttctgtaac tgtgttaatg ccctttgttc 19700 ttccatatgt taggtatgct ggtatagttg aactctgctg actctcctca 19750 gtaaacagtc tctttttatg acaccttatc ctctactgaa ttctctctat 19800 caagaatgac ttggccgggc atgggggctc atgcctgtaa tcccagcatt 19850 ctgggaggcc gaggtgggca gatcacccga ggtcagaagt tcaagaccag 19900 cccggccaac acggtgaaac cctgtctcta tgaaaataca aaaatcagct 19950 gggcgtggtg gcaggtgcct gtaatcccag ctacttggga ggctgaggcg 20000 ggagaatcac ttgaacctga gggggaggtt gcagtaagcc gggatggcac 20050 attgcactcc agactgggtg atggagaaac tccatctcag ggggaaaaaa 20100 aaaaaaaaaa aaagaatgac ttgtcttcct cttagagtgt gaggtctaca 20150 tacaaatatt attcttgtat tcagcaaatg tatgtcatag gcctagtgtg 20200 tgttaggaac tgtgctgtca ccaacaaagt ttagagaggt tataaaactt 20250 gactgtagct ttttagaggt ggaggagtga tttgaaacct aggctgtaat 20300 tccttcctcc tgtgattcct tcctactgtg ttgccttccc ttgaaaattg 20350 catttggggg ccaggtgtgg tggctctcgc ctgtaatccc agcactttgg 20400 gaggctgagg cgggtggatc acctgaggtc aggagttcaa gaccagcctg 20450 gccaacatgg cgaaaccccg tctttactaa aaatacaaaa attagctgga 20500 tgtggtgtgt ggtgacatgc acctatattc ccaggtactc agtaggctga 20550 ggcaagagaa tcacttgaac ccaggaggca gaggctgcag tgagctgaaa 20600 ttgcaccact gcactccagc ctgagtgaca gagtgagact ctgtctcaaa 20650 aaaaaaaaaa agaaaagaaa gaaaattgca tttagttcct gtagactgtg 20700 tgtcaaatgt ctaaatctct tctaaacaaat ggcctaagga ggtgcaaagc 20750 gaagcatcct caccagcatc ctgacttggc agtgaggcat gggaccctgg 20800 agggagtagt ggtaagtgtg actctggaat tcttcctggg ctacttgtca 20850 gtgactggct ccagattgag aggagagccc agaggacaca ggtggctgcc 20900 ccagcctgga ggtgaaagtc ttaaaataaa atgccagatg cctagaccat 20950 tctaaacctt tctgagaagc tgaaatcatc ccttctggaa gcgctctagt 21000 tctaaaagga cagatataca gcaagatctt cctggggcta atatggagtt 21050 tataggcaag taggcctcag aacctttccc tggtagtgat atctgtgggc 21100 aggcacagtt tccacacttt ccagaaattc cagcggaagg agtgagaagg 21150 aggaatctgc ccttgagtga ggaccaaaga aagcagaaat tcctcttggg 21200 aatttttcct ccagagacca aacactactt gggagcttgt ttactgggct 21250 ttaaaagctt gtgaccccca gtcactcttt cttgaccccca aggctttgca 21300 tttctgtggc ttccccactg gacagaagtg gaactgtcat gctgcctgtt 21350 ctggggtctc ccagaggttt ccccatgtcc tctccttgct tctactgccc 21400 cacagaattg gggatctgtg accacatatg gtatagaatt aatgcttgag 21450 aatggtttag ttcagtgatg tcaaataaga ttcactttta tgccacctcc 21500 atcagttgaa ggcccccctg gcccctaaat tggaaaaagat tctgagacag 21550 aatccccgtg ggtacagcgc agggacagta aaggcacgtg tgctgtgatt 21600 tgctatccac tgtgtggatg catccaggaa tatcagaacc ctggaagatt 21650 atttaagggg aagttaggac agcttttttg ccaatccaag ggtgttcttg 21700 aggaagtctg tcttcctgta tggccttcag tttctttcct gtgtaaccat 21750 ggggccaaca cataattccc acagctctat tggcccttgt ctgccaggat 21800 tctctagggt ctgattcgag gtggatcctg gccctttgag gtggcagaat 21850 ctgatcatgg tgctgtttcc ttagatttag gccttgatac ccttggcgag 21900 agcatcctgg gctgagtgac cacctgaggt ttttctggtg attttgtgac 21950 ccatgtaaaa ctttgagctt tgggattatt ctctcaagga aatagtgaca 22000 tttggtgaag agcctgtttg gtgtggctat gtgaggctta gccaagaaaa 22050 tgcaccattt ttattaggag gttaggccat ccgttgccac aaagtgtcag 22100 atgctaggcc tagagcctgg agaaaactta ttttaaaatt gatggggtgc 22150 tggagggggtt ggggggtggt ggctgtagct catgaatcag gtgctaaaacc 22200 tagaaacaaa aggcctcatg tggcagactg tttctgagca cagatgaatg 22250 gatgagcaac tggcgcaact ttgcccagtt ggtccagctt cccacttggc 22300 cacctaggct tgctgtgaag acctcgtctg gcagaaatga gagtgttttt 22350 gccccatctt gatcttaact gtaatttaag actaaaatct tagattctaa 22400 aacatcaaag gcaagatggc tcccagctct gtgagctcag cttctcacct 22450 cttagttgaa caagtgcagt gtgggtcaat acatgattgc tgctcttgct 22500 gccaggaact gtcccagcat agaaaggaat gggacacaat ccctgccgtc 22550 aagattctaa gggaggaagc aggcaggtcg actggtgcct catctctgca 22600 gggctccagc caaggtttgt gaaggatttt gcaggcatat ggagtgggga 22650 ctgattgatc ccgagagggg actggggaaa gctctgaaga ggggatgaca 22700 tttggtttga actccaaaaa atggttgctt tacctgtttc ctgaagtttt 22750 tgaggtggct tataagaaca tataccataa aaaggaccaa tataaattta 22800 aaatcagaaa aagagaaaat gggctgggca tggtggctca tgcctgtaat 22850 cccagcactt tgggaggcca aggtgggtgg atcgtgaggt caggagatcg 22900 agaccatcct gcctggccaa catggtgaaa ccccggctct actaaaaata 22950 caaaaaatta gctgggtgtg gtggcacatg cctgtagtcc cacctacttg 23000 ggaggctgag gcaggagaat cgcttgaaac ctgggaggcg gaggttgcag 23050 tgagctgaga tcgcaccact gcactccagc ctgggcgaca gagtgagact 23100 cctcctcaaa aataaataaa taaagagaaa atggaactta gaaaattaag 23150 aggaagagtg aaaaggtaga tatttagtca ggcacagtgg ctcatgcctg 23200 taatcccaac actttggggag gccaagacag gaaaatctct tgagaccagg 23250 agcttgagac ttgcctggca acatctcagg tgagacctta tctctacaaa 23300 aaaatttaaaa attagctgag ctgtgtggct cgtgactgtg atcccagcta 23350 ctcaggaggc cgagaccaca gcccaggagg atcgcttggg cccagcagtt 23400 tgaggctgca gtgagctggc accactgcaa ttcagcctgg gctacagagc 23450 aagacccagt ttaaaaaaaaa aaaaaaagat attcaaacca tgggtcccaa 23500 cgtagttat atatttgacc atttgcaaaa gctgaaagca aaacatgtta 23550 cacattttca gagaggaaaa tacacagtag ttcctgagtg taagttgttt 23600 ttcttgacct cattcttaaa ttgcttcatg agggtgggag ggaagtggta 23650 gttaataagt gaacctgtaa accagcgttt ctcaaaatgt agtccaggga 23700 attgcatcaa aattgcagtt acctacagtg cttgttaaaa tgcagattcc 23750 tgggcccctg ccccaggctt atcaaatcaa tctggtgagt aggactcaag 23800 aacctgtaaa ttcacatact tctgcagatg attcttcttg cactgcacag 23850 catgaaagcc tctgcaatag acagaaagct accagcattg cgaaagcaac 23900 ttgagtgctt ggcctttgaa ggttgagtgg gactttaatg agggagagag 23950 taaggcatga gaaatggcag ttccactgag gtcagtcagt ggttcattgc 24000 tgacgaagtc acttttaagt catgttttag aagaactacc aagtgtggca 24050 ggtcaggcat gtggcaggac tgtttctgag cacagatgaa tggatgagca 24100 cctggcccca ctgtgcccag ttggtctagc ttcccacttg gccacctacg 24150 gtctgctgtg tggaccttgt ctggcagtct cctttaattt attttttatt 24200 atttttttct ttttgagatg gagtcttgct ttgttgccca ggctagagtg 24250 cagtggcatg atctcggctc actgcagcct ccacttccca ggttccagcg 24300 attctcctgc ctcagcctcc caggtagctg ggatcacagg caagtgccac 24350 cacgcccagc taatttttgt atttttaata gagacatggt tttaccatgt 24400 tggccaggct ggtctcgaac tcctgacctc aggtgatcca cccatctcag 24450 cctcccaaaa tgctggaatt acaggtgtga gccaccgcac ctggcctatt 24500 ttttttcagc aaattctttg tttttctctc tgttcccaaa tgcagggtac 24550 tgagaccaca gatgtattct gtttcctgtt gaaaaaatgt ttctcactta 24600 gctgggtgtg gtagcatgca ctgcagtccc acgggaggct gaggcgagag 24650 gattgcttga gcccaggagt tcgataatca tgccattgca ctctggtctg 24700 ggtaacagag cgagaaactg tctcttaaaa aaaagaaaaa gaaaaagagg 24750 tcctagggaa agaaacaaaat agtggcttgg atggtgagtt ggtggaaaga 24800 acagtgggtg ttgggggtgt tgaacttgtg tttgtgtgtg gtgtacccaa 24850 gacatatcat gtcagcatta agaatagact attcctgttt tctggtcact 24900 gagttgtatg ttttgacatc cttattttgg aagatacttc cttactagga 24950 atgggatagg gagggggtca cctttcccat ctgtgggtca tattttaaaa 25000 tatttattgt tcaagtttaa agatataacc aaaggtataa agaaaaatac 25050 cacaaacatc tgatttaaga aacaaaccag ccgagcgcgg tggctcgtgc 25100 ctgtaatccc agcactgtgg gaggccgagg caggcagatc atgaggtcaa 25150 gagatcgaga ccatcctggc caacatggtg aaaccccgtc tctactgaaa 25200 atacaaaaat taactggtca tggtggtgtg tgcctgtagt cccagctact 25250 cgggaggctg tggcaggaga atcgcttgaa cccaggaggc ggaggttgta 25300 gtgagccaag attgtgccac tgcattctag cctggcgaca gagtgagact 25350 ccgtctcaaa aagaaaaaaa aaagaaagaa atcatttcct acaccttcga 25400 agccttcatg agttagattt tgaaacagtg caaaatgctt cacgtgagaa 25450 tcgagagtcc cttctggtgg ctctccatcc cctgctcttc tgtcaggttt 25500 tcttgtaggt ttatggaaac ctttgttact tgtgcaggtg gcagagaagc 25550 agagaggata gctgcgcgcc acccacacag ctaggattta ttggcgtact 25600 cccacgtgca tggcagccaa gtggacacaa ctctgtgatg aatcctccca 25650 agagaactga ggggccctga tggaggagct gcttctttgc aaagctttcc 25700 ttgactctct tcctgtcccc tagttgattc cccttctgtg ctagttttag 25750 cttattgttt gttacctgtc acacttagca gtactgttgg ctttgctggt 25800 ctccttgact actgggggta aagacctttt gttgttgttg ttgagacaga 25850 gtcttgctct gtcgcccagg ctggagtgca atggcgtgat ttcggctcac 25900 tgcaaccttc acctcccagg ttcaagagat tctcctgcct cagcctccta 25950 agtagctggg attacagcta caccacaccc ggttaatttt tgtattttta 26000 atagagatgg ggtttagtag agatggggtt tcaccatgtt ggccaggctg 26050 gtctcaagcc cctgacctca aggtgacctg cctgtctcag cctcccaaag 26100 tgctgggatt acagacatga gccaccatgc ccagcctcaa agacctcttc 26150 tttacttgct caccctgccg cccactcccc taccaaccct tgcatgccct 26200 ataccacctg gcacatgata catactaact gggtacatgt ttgaatatga 26250 atggatgtgg tgctgtgaat gcttagggga agtgggtgaa atgcttaaga 26300 accaaccttg agtggtctgg gaaggcttcc tgggagggtg gtgtttgagc 26350 taaggccagg cagctgttag atttgttaga ctgaagccct tgcagactta 26400 gagagcttgt gctcttccca gaatgacggg tgagccacgt acagtaaatg 26450 gtgcttctca tttctagccc aagggggcctc aagggggcacc gtgatttcac 26500 gagaatgctg caagcaaatc ttttctcaag ctggggaatt tggtggtaat 26550 gcctggctca gcttgcggtg cgcacctggc ctttggaaga ttggtacaga 26600 gagaagcggc ccatccacat gagcctgtgg aacagcactg gtgggggagc 26650 tgatttgtga agaggggctg tgcagtgtac tgtcaggtct gagacccagg 26700 aagaaattcc agtatcccag ctctcagaat cacagagttc taggcactgc 26750 ctagttccac gtgttcccaa atgtttcctg aatacttgga tttcctgtcc 26800 agagaatttt caaaacaaac ttagaggcct gacccatggc tgccaaggaa 26850 ggattttttt tttaaattaa attttaaaaa tcagtccagc atgaaaatct 26900 atgatgattt catagagaa aggacatttt aatattcaaa gagtaagaag 26950 cacttaatct tggaagaaag ggcattccta tactttgatt acctttagtt 27000 taattaaaaa acacctacat ggtctttact tctgtgattt cattcctggg 27050 ctagtgaaac attgtcacaa taaagcatca ggccaacgct tctttcgacc 27100 cactggccaa tcagttgaca aacagtgact agatgtttca gcctattttg 27150 ctgaggctaa aggattgaac tagtgcttca gccagcatga aaaccagtca 27200 ggagtccgtg ctggtgttgg cttagattag cagggccttt gatgggagggg 27250 catgtatgtg tttgggtttg ctgtgccagg caggggagca gtggaatttg 27300 tctgaattga gctcacacat tgaagttatt gagcgactta catgcaaggc 27350 catgacctgg actcccagcc gagaggccca cgtggcgggg cttgagctgg 27400 gggagccgag gacagcttac atctgctcat ctgcttacgt aaccctgcct 27450 cccagcttcc agagccaaga aaaacacacaa gccagcccag cggggccgag 27500 agcctgtggt agcacacgcc atgcgccgca cagcaagggc gccttggctc 27550 ggcttgaggc ctgtcatgaa gccctcagcc ctctgcctcc tcccagagct 27600 tctccccacc accccaggca gtggctctga aacctggtcg caggtctgca 27650 tgattctgaa cagaggtagt cgttgccttc ctggagtctg agctctctgg 27700 agtttctcac tgggacagag ccaggtgtgt agcagagcat ggtccctgca 27750 gtatggcagg aggtgtgcag ggcattcagg aggcctcctg gctggcactc 27800 gacccaatta gtcattcaac gccaggtctg gggctgctgt ctgttgtctc 27850 aaaggtgtga gctgcaagat ccttagagtt gtggagaaaa aattgccaga 27900 ttggcaagaa gggcaggatt gggggtcaag gtgtctcagt gtgttggaag 27950 catgatgggg gttgtgcaag gggcacagcg agttcagaag ggagcaggag 28000 agtgagaaga ggctgttcag tgataaagct ctgcacagag ccattggagg 28050 agcaagctcc ttgaccatcc ttaaaccagg gtaattttca tttaggttct 28100 gccacacgct cagcagggaa ctcctggaag gcaggatttg tcttgtccat 28150 cctccctccc tacctcaacc cactcctcct tgggctggca cacagtaggt 28200 acccaaag tatcaattga aacaaattga aagtggtctt gatacatatc 28250 acagggcaag tttgcagtta acagacattt cagagtaaag actctctggc 28300 ttggtgctcg atcggcttct gtgggttgtc agcatgctgt ggacagcccc 28350 ggcatgggag cgagtgggcg tgtgtgtgtg tgtatgtgag ggtgagagag 28400 cgttagtgtg tgtgttgggg ttggggagag aggagggggga atagaagatg 28450 gaccacccgg gtatcagctt ctgccctggg gagatggtgg tgtcagttgc 28500 tgagggaatc ctgagaagca ggtctggctg taggtggtga tggtggtggg 28550 gttgcatgag aatccatttg gggcaggttg aatttgaggt gcccatgaca 28600 tatggctagc catgttctgt tggctgtgag gtcaggagag agacatgaga 28650 tggaaacaga ggtttgggaa ctgtcatgtg cttaaaccaa agacctgggt 28700 atagggagag tgagaagaga agggggcaaa gatggacatc caagaaagaa 28750 gctgagaaag cctaggaatt tgaggtaaga ggagacgtag gtaaatgtga 28800 cgcttggtga tcaaggcttc tttccacctc tcctatgctg gacactcacg 28850 tctcctgtct gcttggaaat tcatgctgag ggcagggaag gtgggagcaa 28900 ggatttgtct aaagatcttg ctttggatcc ctgcactcct cctggtttac 28950 caagtgtcac tggacacgtc agggcgttct gagaccttag agagcatcca 29000 gtcctgtccc tgcagtttac aaatgaggaa accagtaccc tgagagtggc 29050 tgtactatcc actctcagga taccaaagat catctggaaa gtcactggtg 29100 gagctggacc ggggcccagg catctcttct cctgtccggg gctcttgact 29150 tcaggaccac ctttctgaaa cccatgatgg ggcaacacca ggacactttc 29200 cagcctgcag gtgtctgtcc cgcggaagcg agccaggcca catgtgaatt 29250 cctgttttct gggtgggttt cagaaggtac gagcaagtcg gcagggtgac 29300 agcccaggtg cttcttgggt tccccaaac gcggttatgt ttagcagcat 29350 cctcagaacc aaaggtgggg tgggggctgc agatgttgtg ggggccctct 29400 gaagtgaaaa gagccctgtg acagatcttt tcttcatgtt tttcacaagt 29450 tcactgtgca gcagggcccc cccagtagcc tttgcccagg gttgggtgtt 29500 gggcagccca ggcctggctg accttgtggg gaagggtgtg aatggtggga 29550 atccccgagg gccctctttg cccgaaagcc ctaagccttg acatcagatg 29600 cccatcagat ggtccatcgg agccctacta cccagcttgc ccagtgagaa 29650 tcatctgggc tccttgttag gtagccattt aggtccttcc caaaatccac 29700 agactctcta agggaagggc ccgagatgct gtacttgtac taacttcctc 29750 aagcaattct tgtgataggt ttgggaaaaa cttgtccagg gtgaccactg 29800 actgagtcct ggtcttctct gaagagcaca gtgcctgctc actttagggc 29850 accctgggag gtgggagctg gctcagcagg cagtcttata agggactgag 29900 cttcaaggcc tctgtccctc caggagggag gtgcatgacc agagagggag 29950 gcctgaggat cttcttccct gccccagagg gtctgctgcc tgagctctgt 30000 gatagcgcag agagtaaaag gatcaagctt gattgaggcc tatctctcaa 30050 tgcgaaagtt tgctagttaa gaggagagtg ggaagggcat ttctggcaaa 30100 gagaaaagtg tggacaggca tggcttaagg gatggggagg gagacagaca 30150 gagctgaggg tgaagggcct tttgctcagc tgtgggcctt ggccttccct 30200 tgtgcaggga cacacagcct tagagccact ggaggtttta gtgggaaagt 30250 aatatggtcg gggctgtatc tcagaagaaa acaaactaat gggaacaggt 30300 cctgtgatgg tggacctggg tcagctacgg agggagggaa gatgtgagat 30350 gtgtactggg gaagggggtg gaagtggcag ctatctggtg agaggaagca 30400 ggcccacagc tttttttctc aagctgttga attcagaagg gcgagtgatt 30450 ccgggagtag ggggtgcttg gagagccacg cgttatgat aaacagggca 30500 ggctgaagcc tgctcactgg ccctgggcgg gttctcacca gcatgtttca 30550 ggttttgatc tgtgcttgtg gttggtgttc ctacctgttc tctaggttcc 30600 ttcctttgtt cttgtggctc atttgcttca caggtgaagc tggttacact 30650 agagtaacag ttcccaaagt gtgttccctg gaaaaaatggt tctgtagcca 30700 aataagcttg ggaaatggtg ggttaaatat aacgaagggg gtttttcgac 30750 tgcacaactt ctcagagcct ttggtgtgtg tcgtgacttt gcagaagcag 30800 gatttaatac gcagcattcc cgttcttatt tgaccacgag acatgttttt 30850 ccattaagca tcttgctggg tctgatgttt tctggaaccc attttgaggc 30900 ggtctggtct gcagagagta tggggagcct gggttcaagc cttggctctt 30950 gactctcagc agagccttga ttccctgtgt tgcctggact gcaccacgtg 31000 taccacatac ccggtatgtg acgttttcct catccctctt cccacctgcc 31050 gttacctcac aatccacaat ctgcacctca tccatttttc ttctgaggca 31100 agcactctct tactaactta cttatctcat ctgcatccat gttcttctag 31150 gccagaaact tggggagtcat ccctccctct ttgttacttc ttcttcctct 31200 ttgttacttt atcccctctg ttactaaaaca ttcttctgtg tttccagcta 31250 tttcttttat tttccctcgg tctcctttgg ggtttctttg cctccatctc 31300 tcccagacct tggttcacct tccatcgagt cccttcctgg gacatgggca 31350 ctcatgccac tcctgctacc ttccacttcg aagctaactc cctccacact 31400 gacgtcccca acatgcatgc atacacacac acacacacac acacacatac 31450 acacacacac acacacactt ccccagttag gctagaatca gagagatgat 31500 gtcagccatt tgtccaaggc cacgcagctg ggaggtcaca gagctaagtc 31550 tcaacctcag gggttttgag aaattgcctt ctcatccgtg atcactgatt 31600 tctacaacag cctgtcagga agtctgggta gaaattactt ccattttaca 31650 gtggagtcag agcggggagg gtcctgggca ggcgagtgct tcacagagtg 31700 accaaccatc taggtttgcc ccacactgaa gggggtttct ggggatggtt 31750 ggtcacccta atgctggatg tggtgcctga tgctgggcag gagggccctc 31800 tccgtggcca cgttgcctcc caggaggaga catttcctct gcagctgcag 31850 ctgcagcctg gccatctgat gcagcctgtg gagcggtggc gagtcctgtg 31900 gcctgctaac ttctccctcc ctccacctct ctagtgggcc ccatgctgat 31950 tgagtttaac atgcctgtgg acctggagct cgtggcaaag cagaacccaa 32000 atgtgaagat gggcggccgc tatgccccca gggactgcgt ctctcctcac 32050 aaggtggcca tcatcattcc attccgcaac cggcaggagc acctcaagta 32100 ctggctatat tatttgcacc cagtcctgca gcgccagcag ctggactatg 32150 gcatctatgt tatcaaccag gtgaggcctg ggaaggtgga atgagagagg 32200 gtgtgtgtgc atgcagatgt gtatcagatg tgtgtgtaat gagggcaggg 32250 gaagggggagt gatttcacag acacctggca cttacagcga ggaaccagcc 32300 ccccagccac caccagtgca gatgaggtaa acgccaaaaca gtgtgcttgc 32350 ctattgctgt caactctata gccaagggaa atgctggagt gttttcgttg 32400 ttctgttttt gttttctgga agtagccttc cagcaagatt gggaaaaaag 32450 acaaccctaa ttattccaaa gtacacactg attattccct ggctttgtgt 32500 agctgtgtat tttcctttta aaaataaaac caccatttag atgtcagact 32550 tttaggtaac ttcaaagttt atccagtcag tcagagcgtg tctcctgggg 32600 cacctggaga cagtgccctt agttcaggtc acatgcctac atgccagccc 32650 ctggtgaaat atctggagaa gtctgattcg tgggccatct gagagttatg 32700 tggactgggc cgagtctgag aaaaagtttc tcactgctcg tctgatccat 32750 atgtgttggg ctttagccct gcttaggaaa gtaatgctaa ggataggtca 32800 actttcatca ccatggcatg gagaatcaga ttgatctaag aggcatcttt 32850 attgaaataa atttttcagt ttatttgagg agcattattt tcccaagagt 32900 ataactttga tatttcaaga ttacccctaa cacttaaatt catgttttta 32950 gactataacc tcctaggtgc aatgacacat ctaacttatc taagcaccca 33000 gtttcattga aattcatttg aagagtctga gtacgcccat ttctacaagg 33050 cccaatgtcc atttcatttc gagataaact ctgctttagg taggaggatt 33100 gttggcagtt tacggcttcc atcaaggtca aggaactctg tgcaccttcc 33150 ctatgacccc aggggaagca ctcgaggact gctgtggcat tgtgctgcat 33200 cacttgctgc agggagattc tgaagaagtg taaggtctca gtcctgccct 33250 gtcccgaagc ctccaaccca cttctggcaa gtgggacctt cccagggaac 33300 aatttgttaa cagacccaaa tatcctgtga ttggatggtg gctgccaaat 33350 gctttggaag ctcagaggaa ggagagagag caatggcttg gaagaaccag 33400 gatataaact aggttctaaa gtctgcaggg agatgggctt ctcagctggg 33450 gccagtgagc agggacctta aggcagaaag gagccttgca tgttcctgga 33500 aattgagatg cccactgggg taggaaagca ccagaagctc tgggaccagg 33550 tgtcagagtt aagcctgtga ggcaggagag agcagaacaa gccctgttac 33600 aaggaaactg aagcaggaga gcaggtggtg ggcaaacccc ttgaggctgt 33650 ttgaattctt cggccaagtg aggtacagac cagggcccta tgaacacctg 33700 caagcaagac agccacgcag ttgtgggtca ccttggaaga atattggaga 33750 atgcaagaga gaacaggtaa atgtcctgca aaatgcgggt cactttaacc 33800 caacacatat tcatttaaga aaagctctgt gattgagaaa catttgtctg 33850 atgccagtta gcacatacca atgacggcaa gattcaggag cctgttatta 33900 aagcagtggc agcgagcacc tggaagaggc ggccaccatc accaggagcc 33950 agcagggatg actaataagc cgtgccagct gcatctcgtt tctctcttga 34000 cagttgctat gccagtagat gagggatgta ctgtggatac aatgctgtca 34050 tatcttattc agcagggcat ctgatagcat ccccaaaatc tgcctgagta 34100 gaagacagac agctgtggtc tgggtgccat ataggtaggt taaaatatat 34150 atttgggcct aggcgcagtg gctcatgcct gtaatcccag cactttggga 34200 ggccaaggca ggcggatcac ttgaagtcag gagttcaaga ccagcctggc 34250 caacatggcg aaaccccgtc tctactaaaa atacaaaaat tagctggaca 34300 tagtggtggg cggctgtaat cccagctact cgggaggctg aggcaggaga 34350 atctcttgaa cccagggaggc agaggttgca gtgagccgag atcatgccac 34400 tgcactccag cctgggcaac agagtgagac tctgtctcaa aaaaataaaa 34450 taaataaata aataaataaa atatatactt gggtaaagag gataaaagag 34500 ttagcgatga tgctgaattt ttgaactgag gtggctgttt tcaaggaaga 34550 ctggagggtg ggatgctacg tctagatatg ttgcagttta ggtgaatgtg 34600 agacttccct gttttgaagt caaatattgg accagtaaaa tctagccatc 34650 agcttaaatt cctatgatac aatttacata ctccccaggc tcaacacagt 34700 agatttctga atgtcctctg ccagctacat gctcctgccc acctcaatcc 34750 gagtagatgg aacaactaac caagccagct cagaccggtg gcacagctgt 34800 gctggctaac actgggcacc acctaagaga gtgcttctcc aaaagtgtgc 34850 ttccccaaat ggagcgaaat acgcttgagg aatgttgggt tgaaccatgt 34900 aaagcaggtc tcattcccgc agagcctttg gtaccccggt gtacactgta 34950 accccagaag tgtttcctga gcttgcctga cgagacaact tttccaagaa 35000 ccgtctcaag tgatgagtgt tttgtgagtc acactttggg gaaagcgggc 35050 ctaagttagc atctcctccc agctgcctcc ctgctttccc tggaacacta 35100 ggaactgccc gtcctccctc cctccctcct cttcccactt cacaacttag 35150 catcaggaat attttagttt tggtttttca aacatatata cctccttttt 35200 tcttatcttg tcaatatcat ctttttttt tctttgcttt tcctcatact 35250 tttttttctc ttcatccttt ccttctccaa gggttaactt tccaccttag 35300 gagaatcttt tctgcttttt ctcccacttc cccagctact ctcttatcat 35350 ctgctccaat ctcaccctaa ttgatcattt tgggaaaata tggtcagagt 35400 ccagataact aagttgagaa atgcttaaac tctgccatac ctttccagta 35450 aagaatatta cctaataaat aataaaatgg taatgggaaa cctgaaccct 35500 gaaaaaaaag aggtggaagg agaaacatt ggagcacatc ctgtctacaa 35550 attaggaact gcctgtgtta tctgttttat ggttatattc tagaagaaga 35600 aagggatttt gtagcacctg gttttgacct ttctgcactg tttgttgagc 35650 aaataaacct tatgggctgt tagccctctt tatagcctct cagcttatcc 35700 ctggcccaga caccctgctg tcattttgac ttttcattcc cacacacaca 35750 tacacatgca cacacatgta cacacacaca cataccattt aagattagac 35800 agaagtaatg ctcaaaatgg agtggcttct gagacattta gtccaagggt 35850 tcccaaacag gcttttcagt atcagatttc tttctgcccc attgaaatgc 35900 tacacaacct tccgcttaca gcaggtcaca agggtttcat tctacttgaa 35950 gtaggggcca tgtcccattt ccacttcctt ggcttcccat tcagtcactg 36000 ctaggatttg cctagacccc tgaggccaga caatgtagaa acttctgctc 36050 catgtcacag gtgaggaaac aggctcagag agggacaggc tccgaaagtc 36100 acatagacaa cagtagggct gcggctcaaa ccccagcgtc tgactccagg 36150 tttagtgcct tctcagggca tcagtgacac tcctcatggc cagggtgccc 36200 ccagtgttgc tcacagtctg gtatccaggg ctgagagtgt gctgtgtgct 36250 cagactgcct gggttcagtc ctggcactgc cactttacag tcagtgacct 36300 caggcaggtt acttaagctc tgcaggcctc agtttcctcc ttggtgggga 36350 gggttatgag gcatccttct catggtaaac cttcagtaaa taccagccgt 36400 tactaggagg gtccactcct gcctctccac tctccattca tcctgcctgt 36450 ttcctctgcc tgcttcctct gcctgcttct gtggtggtga attcttcatg 36500 gctcccaccg cctcctgctg cacccccact cagggcccgc atcaggaccc 36550 ttcctcctat tggtttgaac tccttggagt cagagggtaa tggatagtgg 36600 agtgagccag gtggcagaat ctcagaggcc atcccgggcc tataagcctc 36650 ttcaaaatag ggccacgtat caagctttac acacaggagt gaactttcac 36700 aagttgttat gactcatact ctgtctatag taagctgtta accactccca 36750 tttggcttat gcctctgtaa ttattgtact aacttatatc ttaaaataag 36800 gatattgaag gaatgagccg ggagaggctt tcctggttga gatatagaag 36850 aacaagagtt gctctttttc cttaaggtct ctcctcccac ccctgacctt 36900 agctcaccag catgggagaa tactatttga ctccttgtac tctgagacgt 36950 ggatttcaag atatagcatt ccaacttcaa cggcagcaag aaaagaagca 37000 acagaaggag aagacatcat agcaaacagg gatgcatgct gcatttccta 37050 atactcaaac ccggaaacga gacttcactc aaggtgaagg gagggcaggt 37100 caccacctgg tagcactagc cctaaattaa ggaatgcaga atgtttgtgg 37150 gattgcccat cataaaaatt acaaaatgag taaggaatgc aggcacagct 37200 ggccaggtgg gtttgtcaca accatggcag ccctttgccc cacagccagt 37250 acacagaact ggtctctcca attccgattg catatcttct ggcacctctg 37300 ttcctctccc tcagctgccc aggatttttc tggttctgac catgttactt 37350 cctcttttaa acctgttagc atttcacgac tgcctacagg caacggtcta 37400 aatggtcgga aggcccaagc ttagcatccg agaccctgac ctacctccag 37450 ccacttcctc ctcctctcca cttcactgga ctccccatct ccacccagac 37500 acctctgttc tcccctctgt gtgcctttgc ttatgctgtc ccctgtgttc 37550 ctagtgtgtc tctggctatc ttttaagctt ccctccccaa cctcattagt 37600 tctgtggagc ccctggaata gagctgactt ctccttccct gctgctccca 37650 ggctgctcag aactttctgg aaaggggatga ttatctgagt tccagcctca 37700 ccccagcccc cggactctga gtccctcatg tctgcctccc ttctttctct 37750 ctgaccacac agctggtaca tagtcagtac agacgcagtc agtgagtgga 37800 gcacggggct tctctccagg attcctgccc ctttgtttat ccctagtctc 37850 aggactccct actcctggtc ttctgcctaa atctgtgcct cttggaagtg 37900 aagcctccgt tcccagtggg gccaggtcct gacccttggg aacttgcagg 37950 atccctccct tgggcctctc cccgaagctt ccagctcaat gctgaccaga 38000 gcacaggctg cctgtgacag tccttggggt gacctccctt atcaggaaaa 38050 atgcagaaaa cctattaata ccttagcctt gtgattgtta atggtcacaa 38100 aactccttta gggtcctttg gactcagcac ctttatggtc tcactttgaa 38150 ttttgaacct cccacctccc cccatccccc agagtaaggc aaatggtctt 38200 ctgattgttc ctgcagaggg aaggctccac aggtaagcac acgatggcca 38250 ggaagcagag ctggagcctg cctgaaaggc tgtggagaaa tggaggggagg 38300 gctgccctga ggactctgtc tggctttgaa gttttctact gtttcctttt 38350 cttctgtgca ctgttttagg atgatggggt gatagttcca ggctggttga 38400 ggatggattt ggagacagtc ctttgtaccc tcagtgagca agagtatctg 38450 tcaccctacc tcagcagttg tctctgtcac tggtccaagc agctggttcc 38500 tacacaaggt caagatcaac tggggagaag cagactcctg ggtctatccc 38550 attagtgagg acagctgcct gggcttatgg cctcattggt ttggtttcta 38600 tcttgatcat ctctaccatc cccccatccc ggccttccat tttctacctc 38650 agctgtcagt gcacagattg atgtgtgtgg gaacggagct tgggaggagt 38700 ggggtagggc tggtcctgtc ctgtagcctc cccttccttc gggcacttgg 38750 accctttgga gcttgccggg gtggggaatg ggagtgggaa ggccagggag 38800 tgtctctgca ccatcactgt ttgagtgttg cccctttgct gtgtgcccca 38850 cctagtctat gtgtgtctct gttctctggg gactcaattt gctggtgaat 38900 tgcttccatg gacattgttc tgggaaatgc cattttttct gctcacccat 38950 gactctgtga caaggaatga cagcttatta ggaatttgtt tttgcattgg 39000 aacagtggtc atcagaatgg gccccttttc ccttgcagct ttgacatttg 39050 cctctctttt cctcacctct ctcccttgca tccacccttt tctctttttc 39100 ttcttttttg ttttccttct agcaggggcc ttttaccttt acttgttaat 39150 cctgtttgta gcaaagcaag tggaagggagg agttcctctc tgatctgctt 39200 cttatctcc acctaccttc tcttctgtac tttccgcctc ctagagagag 39250 agagagagag aggaatgccg acctaactac cgctgccact gctgctgcca 39300 ccaccgctgc caccaaccacc ctggtaatgt tcacatgtcc tcaaatcaac 39350 ccagagccag ggccctgctg gtcaggggga ggctatgtaa ataatcccat 39400 gagtgtgcca tcctcaggcc ctggggtctc ctaggcaaga ccagggcctc 39450 tgtgggctct ctcggaaatg ctgaggttgc tggaagccag cccgtcatac 39500 agggtctgag agtttaactt cttttaaatt aaaccacagt tgagctcatg 39550 ctgtgtgtgt ataaactttt gtatcctgct ttttccttaa attctttatc 39600 atcagcatct tcccatgtta tttcatagtc ttcatcatca tcactttcca 39650 taccttcata gtagttgatc gtagaattcc atcataatta acttgtcttt 39700 tctctcttag aagtccctta ggtaatgtcc aattttccgt gagtgtaagt 39750 aataccataa tgaacatctt ggagtctgaa gtttatctg tgttggtttg 39800 ttccacatt aggatcattt tcccaggcta gattttcaga tgtgggatta 39850 tgggttcaga tatggtttac acatttttat agttcttaat acagatggcc 39900 aaattgcttt ctgaaagaga agcttttctt aagtattttt ctccaacttg 39950 tatcttaaac atcctgaaca tgcttagcac cactgtcttg atatatctgc 40000 ggaaagccac gtctccactt ttcagtgtgt cgggccctgg gagaggcagg 40050 catcctgcgc tggctccttg gagctgggtt taaaattgtc tcctctggct 40100 gggcgtggtg gctcacacct gtaatcccag tactttggga ggccgaggtg 40150 ggcggatcac taggtcagga gatcgagacc atcctggcta acatggtgaa 40200 accccgtctc tactaaaaat acaaaaaatt agccgggcgt ggtggcgggc 40250 acttgaaaag tcccagctac tcgggaggct gaggcaggag aatgatatga 40300 acccgggagg cggagcttgc agtgagccga gatcgcgcca ctgcactcca 40350 gcctgggcga cagagtgaga ctccatttta aaaaaacaaa caaacaaaac 40400 aaaaaaaacaa acaaaacaaaa actgtctctt ctgtgctcac ttcacccaga 40450 atccctgttg ggctcttcaa ggagctcagt tctctctgaa agcaacttta 40500 tagcctcagt ccagtctgtg ttcctgtgtg gcaggggtca agggtatgct 40550 cactcttgag agtggtgtct ttggttgacc aagaaccact cccatagcct 40600 ggtccctaac ccttgaaggc ccatctctct cactcactgg ggtgaagagt 40650 ttaaatctca gatccaagtt ttgttgagag ctctgagcta ccatattgct 40700 atggttaaca atagttaaca atgttaacaa tggttaacta tggttaacaa 40750 tagttaacaa tgtttaacaa ctagagccca gctgggtgtg gtggcatgtg 40800 ctaacagtcc cagcttctca agaggctgag gtgagaagat tgctggagtc 40850 caggagctca aggccagcct gggcaacatg gcgagaccct gtctcccctg 40900 caaaaaaaca acaacaaacaa aagcaaaact agagcccaac tgctgtgaac 40950 tcatggctga gtagatatta ttagccctcc acaaactcag catttgtata 41000 atcccaggct gtttccagta attctctggg gatcatctcc cagcctgtcc 41050 actgttccag gatccacact taggcctata ggaatgcccc gtcagagctt 41100 ctgctgccgc tgatctgtta ctgtttcatg caacccactc ggcctagttc 41150 cttcctctta ctgtctcagt gggcacagaa aagcatacag agggtgtttc 41200 agcaaacatt gccactggct gcagacctgc ccccggatct gtcctgttga 41250 gagcttagtg ctgcgttctt gcatggtggg gaggggtgtg gctctgtgat 41300 gagccagggc atgtgtatag gagcaacagt gtctctctta tcacgtagaa 41350 gttctgactc attgcgagtc ttggctttgg gttaatggtt ccagccatgt 41400 tgctgctgtg tcttttggtg caggagaggc tgggcacagt tggtccctaa 41450 gccattatgg ataagggatg tgtctgctga tatacacaca tggacctgac 41500 atccagggaa ggcagggtga ttggacagaa cagttcttcc agaagctgtt 41550 ggaacttgga caagagtggc ccttggcttt ctgtagttgg tcatctgtcc 41600 cctgttgcaa tcaggggaag gccacacttg ccttccttaa ccacagttag 41650 gattttcttg gggattagac cagattctag cacctgtcct gaacctctcg 41700 ccccgcccct acaaaggctg cttgcaagtg tagtgcacat acacagggag 41750 caggtggggc atggaagtgg aagtggagcc cctgcctttg gcccttgggg 41800 gaggcactgt ctgcttaccc acggttgttg cctcatagga atcatacaac 41850 agcttcctaa ctggtctcct tgccttcagt tggattgggg cacaaatccc 41900 tccttgacat ataaaccatg gtttaaggct ccctgtggcc taaataaaga 41950 taaagcttaa gtatcttaac aagcacctaa cccttctccc cagcctcggt 42000 gatttggctc atcgctgcct tcatgtttca ttctggcttc actcattcgg 42050 aatttcttgt agttccttgg ctgttctctt ttccttaccg cctttacaaa 42100 tgctctcacc atgcatgctt ttctctgctc ctacagatgc cttctctccc 42150 agcaccgcct ccagagtcta tgtctggtcg attctgtctg ctgtctccag 42200 tccccatctt gtggcagtct ctgctcaatc atttggggat tttatatgtt 42250 ttctggcctt tcttttgggg gcctgtcttc tccttctaaa agcagccagt 42300 tgacctagaa ggaagggata actgtaactc ttgtctacca acataagatt 42350 aggcccacc tttaaaagct gcgtctttga aagggacacc tgcacccagc 42400 atgctggctt ctcttcacca agcgtgactt cctacgcatt tcacaggcct 42450 ccagaggtcc ccctgactct cttctgctgt gagaaactct aatcatgtaa 42500 gccacaggct aattcccttg agccttaaat gtttttagta atttcccatt 42550 catcagagaa gcaggatttg ggaggaattt tgaagcaaac actacagaag 42600 gcagagtctc caggtaggat atctaagaga catttggaat ggtctgactg 42650 ttcaagatgg atgggaaagc ctcttcctgt aatgatagta gccaaacattt 42700 gttgtcaggc agtggggccc catttttgag atggggtctc tgtcacccag 42750 gttggagtgc ggtggtgctg tcatggctca ctgcaacctc agcctccccg 42800 ggctgggtct tcttaattct gaaaaaccca gcttttaaag ggtggaccta 42850 atcttatgtt ggtagacaat gttgtctcat ttaatacaat gcacatgctc 42900 tccccataac acaaaagagg gaactgaggc ctggaggtgt gatgtacccc 42950 aagtcacata gctaataaat aaagaagcca gcattcctgg gattaaaaat 43000 gcatgtgtct gtcactgtgg tgtatttggt gcttgatcaa tgtttacttg 43050 agcaaatgga ggggcagagg taccgatgag tgtgctcagt gaggagggca 43100 ggagtgaagc tgggcgtctt cccgcctctt gtgagtggtg gggcttggtg 43150 agcttgccag ggcctgtctt tcttatcaaa gaaggtgtgt gccccagtgt 43200 tacagcattt cacccaaagc agcctagaaa atgcttgact tttctgtcat 43250 tccgggggagg acactttcct cctccactgt tctgctggcc tggtgtaccc 43300 acggcccctg atagatgata gcacctgcta aagtgcacca tgcccttccg 43350 tctcactgca tcccacagat gaggccaggc tgggatgagg gagaaaggga 43400 gggatatata gttcaggtta ttttggaaaa ctgcctgacc aattttaagt 43450 ctgggccgga cactggggca tctcaccacg ttgaaagggc cgtggcaccc 43500 cgggcggtga aaggggctgg aaccaggtct gcttcttggg cttctcctcc 43550 agggtgccat tgctcatggg ccttggctgc agaggtgctc attcgtggtt 43600 ccaaaattcc aattcctggg agaggaaaaaa tgcttagttc agtctcagtt 43650 aggcctctgc ttagatcaaa cagccaaggc cagtaggccc agtcctatgg 43700 tagagacatg gcctcaaaga gccctctgct gcagttgttg gggagtgtac 43750 caagagaagg gagcattgtc ctgggctggg cagccctggg ggtctagtgc 43800 atagatgtag aaaggctctg ttggtatacc tccctttgct tgttggaaag 43850 tgctcaacgg ggctgaattg tgtttgacag tgtaagtctg ggctggggtg 43900 agggttgtta caagatgtc aagatgatta aatgaaatgc catttgaaac 43950 acttatccat gccttgtgta tggtatcccc accagtgaat attcacagta 44000 tattataata attccaaacaa cttcataatt ttcatatgca atttctaaac 44050 tttgaacttt tttttttttt tttttttttt tgagacagtg tctcgctctg 44100 ttgcccaggc tggagtgcag tggcgcaatc ttggctcact gcaacctcca 44150 cctcccggct tcaagtgatt ctcctgcctc agcctcctga gtagctagga 44200 atccaggcgc ccgccaccac acccagctaa tttttgtatt tttagtagag 44250 acgggctttc gccatgttgg ccaggctggt ctcaaactcc tgacctgagg 44300 tgatccaccg ccttggcctt ccaaagtgct aggattacat acgtgagcca 44350 ctgtgcccgg caattttttg tgtttttagt agagatgggg tttcaccatg 44400 ttggccaggc tggtctcgaa ctcctgacct caagtgatct gcccgcctca 44450 gcctccctaa tgctgggatt acaggtgtga gccaccacgc ccagcctaaa 44500 ctttgaattt ctttgaaccc atgacttaca cagaattagc tgaacgcaga 44550 attccaaatc aactcagcct gtgggacagc caaaaaacac agtgtgcctt 44600 tgggctcctt cactcaccac gcggggttag aaaactttgt cagaggcttt 44650 aaaaaaggag ctcttgtgtg taaaatgttt ccttgattct ctttctggtg 44700 cctctctttc tctaagtggt ttgcttcccc aagttccccca cctgagtctg 44750 ggtggctgtg gcacatctgt gcattctgta cgcacacagg cagccttttg 44800 gagtgccagt ttccaggtct tggttttat tatttattta tttattttt 44850 tgagatgggg gtctcactct gccgcccagg ctggagtgca gtggtgccgt 44900 catggctcac tgcaacctca acctccctgg gatcagttga gcctcctacc 44950 tcagcctcca gagtactagg gaccaccatg cctggcaaat ttttgtaatt 45000 ttttgtagag gcagagtctc accatgttgc tcaggctggt ctcgagctcc 45050 tagactcaag tgatctgccc accttggcct cccaagtgtt aggattacaa 45100 gtgtgagcca ccatgcccag cccaggtcat cttttgaggg catggagaga 45150 agactttgag catcccactt ttgagattgt gtaccagtcg caagccccta 45200 tgacacactt tttccccaaa gtagagggct ctgactatgt tgatcccaag 45250 agagatggga aagagcattg aatgaggatt ccaaagtatt gggccttagt 45300 tcgtttcctc atgttggtgt tgtgaagatt ctggttagga taacagcatg 45350 tgtgcaggag gctttgtgaa ctgctgagag tgaggcgtgg caatgtcagt 45400 gctaggtttg tccttactaa cctggggcca tgggaattga taagaccaga 45450 ttcccaactc taccccacaa tgtgatccct gtggtgaccc ctcacagggc 45500 tctttggtcg agcttcccag aagggatcac catctgccat tgtatgttga 45550 accccattca ttcattcatt cattcagcca accagcaact atttgttgag 45600 ctcttattgt gtgagaagca gtcttcaagg aactgggtga ataaaaaaaa 45650 caaaacatcc taaccttcat tgagcttaca ttcttactga aagaaaacaa 45700 ataaaacata catgtaatcc tagcactttg ggaggccaag gcaggcggat 45750 cacttgaggt caggaatttg aaaccagcct ggccaacgtg aaacccatct 45800 ctactgaaaa ttaaaaaaaa aaaaaaaaaaa aagccgggca tggtggcaca 45850 tgcctgtaat cccagctact cgcgaggcta aggcaggaga atcgcttgaa 45900 tcctggaggc agaggttgca gtgagccaag atcataccat tatactccag 45950 cctcagtgat gaagcaagac tccatctcaa aaataaaaaa taaaaataaa 46000 aatatgcatt ccctttgcac cagcacactt ggtgcctggg gacctcgtgg 46050 ttggcaccct gaagcaggtg tccctcttct gtcttgcaca ccttgcttct 46100 gtcctggtgt gtatggcatg gccttctgcc ctccatggtg agcactgtga 46150 gggcagaggt tgagttgggt ttgctgtatt tctcaggtgc ctaggtttgt 46200 gcttgacagg tagatggaag gcacacaatg tggtcatcaa acctcagtca 46250 accatataag gaaggtagaa gtgaaaagtc ccataggtac ccaactaatg 46300 tcaccagttt cctggatacc tttcctggag tttatttata gtgtgtataa 46350 ataaatgatg tatgtgttta aatgcctttt tcacctttcc ttttagagct 46400 gcctcttttt aacagttcca ttccattgta tggatgtact atgatttatt 46450 gaaccagttc cctactgatt attctgtttt ttgcagtctt ttgttatgat 46500 gaacattcca cagtgacaat gttgttcata gtcattcaca cacatgcaag 46550 tccttctgca ggatatattt ctagagggga attgctgact cagaggtttt 46600 ggtactctgt gttgattgta gagtgacggc agaaaagtga ggcccaagag 46650 tttcctagtg accatgtgta gtggacaagt caccagtccc tgtgagtgtt 46700 tggcccaaag gctttaaggc atttgatatc actgtttttg tttctgcacc 46750 aggcgggaga cactatattc aatcgtgcta agctcctcaa tgttggcttt 46800 caagaagcct tgaaggacta tgactacacc tgctttgtgt ttagtgacgt 46850 ggacctcatt ccaatgaatg accataatgc gtacaggtgt ttttcacagc 46900 cacggcacat ttccgttgca atggataagt ttggattcag gtaagagata 46950 ctcagtcaga atctgtggta aacatgtctc tctcatgtgt tgactaggaa 47000 atgcagtcct ggcagctcaa gagtgcctct ttaagctctg gagcagaatg 47050 cctcctctga gaaatgggtg ctttgtatta gttgagatgg aaagaagaga 47100 ccagaaatgc ctgtagtctc tgcacatcca gacaaaaaca aattttcccc 47150 cctttttttt ttttgtttgt tttttgagac agggtctggc tctgtcaccc 47200 aggctggagt gcagtgccgt gatcttggct caccgcaacc tctgcctccc 47250 gggttcatgc catcctgtca cctcagcctc ctgagtagct gggactacaa 47300 acacttgcca ccatgcgcag ctaatttttg tatattttgt agagatgggg 47350 ttttgctgta ttgcccagtc tggtctcgaa ctcctgagct caagcaatcc 47400 atctgccttg gcctctcgaa gtgctggatt ataggcatgt ggcaccatgc 47450 ctggcctaag aacagttttt agcatttggg aggggctctc atctttaagc 47500 tccaaatgat actgtatttt cttgcttttt tctttctctt gccccacaag 47550 ttttggaaag taaattggaa tagttttccc ccactgaatt atttagcttg 47600 tatacctcag cagatgttcc ttggcctgtt ttgttttgtt tttgagacag 47650 ggtcttgctc tgtcacccag gctggagtgc agtgacacaa tcatggctca 47700 ctgcagcctt gactgcctgg gctcaatcca tcctgcagcc tcagcctcct 47750 gagtagttgg gactacaggc atgagccagc atgtccagct aattttttat 47800 ttttagtgga gatgaggtct ggctatgttg cccaagctgg gcttgaactc 47850 ttgggctcaa gtgatcctct cacctcagcc ttccaaagca ttgggattac 47900 aggtgtgaac cactgctccc gcccttggcc ctataagaag gaatgtgatt 47950 ctgttttcca gcagggcaca aacttctgct taaatacaaa gcccaaattt 48000 ttccaccaaa atgcccctag tgaagtggcc agcccagatg cccgactagc 48050 gtattatcca aagcatattg tcattggtgg aaaatggcct tatagtccat 48100 tgttttgtct taaaagtaaa tatataaata aacttgtata ttgtttccta 48150 attccgtgtt tatattaaca taaaagtgtt ttaaattacc tgtcagtggc 48200 caggtgcagt ggctcgtgcc tgtaatcgca gcactttggg aggccgaggc 48250 gggcagatca cctgaggtca ggagttcgag accagcctga ccagcatggt 48300 gaaaccctgt ctctactaaa aatacaaaaa ttagccaggt gtggtggcag 48350 gtgcctgtaa tcccagctac tcgggaagct gaggcaggag aattgcttga 48400 acccgggagg cagaggttgc agtgagttga gatcgcgcca ttgaacttca 48450 acttgggcaa cagagcaaga ctctgtctca gagaaagaaa aaaaaaaacc 48500 tatcagttga ataacaaaac cctttccttc cttgctttaa gtgaatctga 48550 agatccagga gctgtgctgc aggtaccctc tatgttgggt acccctggtt 48600 taggctgact agtacagtgt ggttggctca tgtagacagc agacccttta 48650 ttttagatac aacttttttt ctttttcttt tatttttttt gagacagagt 48700 cttgcttgtc acccagcctg gagtgcagtg gcgtgatcat ggctcactat 48750 agccttaaac tccctggctc aagtgatcct ctcacctcgg ctttcctagt 48800 agctgggacc acaggtgtgg gccagcaccc ctggctgatt taaaaaaaaa 48850 aaaatttttt tttttagaga tgtctcacta tgttacccag gctggtcttg 48900 aactcctggg ggctcaagca atcctcctgc tttgacctcc caaagtgctg 48950 ggatgacagg catgaactac tgcacctgct gagatgcaac agctttctgt 49000 cagactcatt ttattctcat catttcttcc tgtcctccct tgctgggagc 49050 atgagagctg tgatgggaat ataggaatgt atgaagtcct tctcccagat 49100 caaaaatcct aacttcttgt cttaaaggga ggaaaatttg aatgtaacct 49150 tacttttaga ctcttcagaa atccttctat acccttccgt ccccgctttc 49200 acccttcctc cctctccgtg tgtgtatctt cttctcttga aacacacagg 49250 tttataccct gacccctctt gattcatccc ttgaagcaca gtggtgaaca 49300 aggaaggggc ccgtgatgcc ctaattcttt gccacagcac catgtttgtt 49350 tcacaaggag cctggcaggt ttgggcttgg ggcagatagg ggagagaaag 49400 cagcagagac agcaaaacca aatcatgtca gcttggcatg tacttccctc 49450 tgaaatagct aagaatccat ttctgtaaaa gcactgatta tcagaaaacc 49500 ttatggcct ggccaccttt ggttcaaacc ctcacattaa taatgtggac 49550 agtagtatga ggtgtgccaa aggtggatga ctcagcacct aagtgatgac 49600 acctaattac gaataggttc attaaagcag accccctggg gacctttgct 49650 tgaggatcct tacagtcaga attcctgaat atatttgaaa ataataattg 49700 catctttat ttcatatgtt ctgtatggtt tggctgactt ccccctcaaa 49750 gtctgagtta gagttttcct taatttatgt gatgggtttg gtctttttgg 49800 attccagaaa gagctgggtg tggtttggag ctgcactcag agtcacacaa 49850 aaccacagcc tttagagaac ccacaggaag gctttggggc acgtcctgat 49900 tcttgacatt tctcatcagt gctgactttg tatcccttag gagttcacaa 49950 ttcataacca ctgaaatatt aaaatacaaa aagttttgga aggatgagag 50000 cccagatgct ctactacttg aaaatatgtt aaaacataag ttcatcatta 50050 tacattttgc taaatcagga taaagtctga agtttcaaag aagttttatt 50100 ttagcaaatt ttcagaaaca ctgcctcaac tgttagggcc agtgttctag 50150 tcagtatgcc tttggaagca tgaaagctgg attggtcgat aggatgggtg 50200 tggaaggggg gctgtgactg ggtgggtaca gagaggctct gaaacaatct 50250 cagattccag gagttcctgg ataaggactt catgtgcggg aacagagcac 50300 aggagaagca gattcctgag ccactcagga agaactgggc ctaggcctgc 50350 tcttgtcact gactggcttt ctacataacc acagaaacag cactgtgttg 50400 tagaaagagg aagatcatac tttttgatat ctgtgtctaa tttaaggtca 50450 tctgagccct gatagaaaag caaaacagac aaaacccttg taactgctcc 50500 ctcccacccc acccaccatc aaaaaagctt tagagaggct ggacatggtg 50550 gctcttgcct gtgatcccag cactttggga ggctaaggtg ggtggatcac 50600 ctgaggtcag gagttcgaga ccagcctgac caatatggtg aaaccccatc 50650 tgtactaaaa atacaaaaat tagccaggtg tggtggcaca cgcctgtagt 50700 cccagctact tgggaggctg agacaggaga attacttgaa aacctgggag 50750 gcggaggttg cagtgagccg agatcacgcc attgtactcc agcctgggct 50800 acagagcgag actccttcaa aaaaaaaaaaa aaaaaaagat ccggtttggt 50850 gtcttacaac tgtaatccca gcactttggg aggccgaggc cggtggatca 50900 cgaggttaag agatcaagac catcctgacc aacatggtga aaccctgtct 50950 ctactaaaaa ttagctgggc gtggtggcag gcgcctgtag tcccagctcc 51000 tcaggaggct gaggcagaag aatcgcttga acccgggagg cggaagttgc 51050 agtgagccta gatcgcgccc ctgcactcca gcctggcaac agagcaagac 51100 tacgtctcaa aaaaaaaata aataaaaact ctagagaagc aaaaagaata 51150 actttaaaag tgtttatgtt ctcagcaagc tttatttgg ggatgtcaga 51200 acttaactaa ccactgctcc ttctgtgtgt atgtttttcc tccagcctac 51250 cttatgttca gtattttgga ggtgtctctg ctctaagtaa acaacagttt 51300 ctaaccatca atggatttcc taataattat tggggctggg gaggagaaga 51350 tgatgacatt tttaacaggt aatggtcata acttagatat ctttctcctc 51400 tgtcaacctt cacttccagt tttttaacca atgcttggtt gttcccccaag 51450 gactgaccct cagatgggat gcacccctag tcagcccaca ttcttaggtg 51500 tggcttccta caggtcctgc aggtgctaaa agggatctgt aggaaaaatga 51550 gtttctgaga tttttgtatt ggcctggaaaa aatgtcaaat gggaaccaag 51600 tgacggggca agtttacttt gacttgctgc atgccgtttt gtactcaagg 51650 agtaaaccaa tgtcctttgt aaaaatccct cctttcatta tggtcccctt 51700 tcactgtgaa acaagtttcc ttgagcagaa tcctaactgt cttcacagaa 51750 gctttgtgtt atatttttat tttggagtat tttcacatat acaaaagaga 51800 tactgtagta taataaacct ttgaggacct atccagcccc agcaaccatt 51850 atggcctggt cagttctgtc ccatccacat cctggggctc tttttaagct 51900 ggtaaatcat tatgatgtgg gttgtcattt acagtggtaa aaaacatcta 51950 tcagtagcat ttgaaagaac attctgctca gtcctctggc tgtagaggct 52000 tcaaccccac cagccaccga tgagcacctt ctccctccag gagccagtct 52050 gagctcatta ctgagtttaa tatcagaata caccctggtg cagcctttct 52100 aaattgcagt accagttaac agaaggtgtc tgtcagagca acacccaagt 52150 cattcaagtt accattgtgt gcaaacttaa cagagaccca cgtcttcaat 52200 ataagccttg aaggaaactc cagttttagt atgtagatgg ggtatcaagt 52250 gtgtgcacat tgaacatctg ctgcatacag agcactgtgc caggcaggcc 52300 caggacactg aaaacctgga catagggtcc agacagaagc aagcctgctt 52350 ccacagaggc actcctgggc agacactctg gactgatatg acagtgtgca 52400 gggccgacag gataccacag gtctgaatgg tcagaacagc tggggagggga 52450 gggagcatcc gcaggcatct agtcccatgc taacgcagtg gcactagaag 52500 gatgggtggt gtgtggagca actttcttga aagataaagg acctaacact 52550 ttctatgcac cacttactgt gtgccaggca aggccaggaa tgtttaagtg 52600 gtctgggatc agccagttct gcctcttaac taactttgct gtcctgctct 52650 ccaggctttc attttggtcc tcattccttt tccttggacc aacacagaat 52700 cctccaccct gttctggctg cctctagtct tgttctcagc cctccatttg 52750 tttttttctg ccttttccca catgttctga agccctccat tcgtatacta 52800 ctttccagag acttccccat ggctaaaagc attttggaaa tactgtatat 52850 taggcccctt tcagatactg gcaaccgttt gtgggatgct ctgagaaggc 52900 ctctgtgact tagcctggcc cttttcagcc catcacctgc cacgtcctac 52950 cccagaccct tgtcaccagt ccccaggagc ttacgttgct ccctgagggc 53000 actaggcttg ctctcacttc catgcctttg cctgtgccat cctggctgcc 53050 caaaatgcta tggcagatac ctgttcatcc tcaactgggc tctgcctagg 53100 cttgctccag cagaggttac aaactctatg cttcttcctc tgtgtctcca 53150 acctcatctt cctcttctca cctccatcct ggccctaaag gccctatgtt 53200 tgaagcattc acactgtata ttctgtgggg cacacggccc cagtgtctgg 53250 cacatggtag tcaacaccac aaaccgcaga accagttgta aaaggacatg 53300 gagtcggaat gtgagtttta accagggtca tgctgggctg ggttctggca 53350 tgatgctggg ttgtgggctg agtgagaaca gcaagggtga tggtggatgg 53400 agcaacagtc ttgcagccgg ggctctcagg ccaagtgtat ggcagctctg 53450 tgataatgac tttcccttta ctctttgcag attagttttt agaggcatgt 53500 ctatatctcg cccaaatgct gtggtcggga ggtgtcgcat gatccgccac 53550 tcaagagaca agaaaaatga acccagtcct cagaggtgca ttctttgttt 53600 attcatactc cttccccctt taggatgagg taggctgcag gtccgaggct 53650 ctgggcctag agggaaattg aggtggtcag gttacagtgg agagggagga 53700 ggaagtacgt gtgatgattt cttcttaaga tttttgtttt aagacaatct 53750 ccttgtgctc ttttccttgt aggtttgacc gaattgcaca cacaaaggag 53800 acaatgctct ctgatggttt gaactcactc acctaccagg tgctggatgt 53850 acagagatac ccattgtata cccaaatcac agtggacatc gggacaccga 53900 gctagcgttt tggtacacgg ataagagacc tgaaattagc cagggacctc 53950 tgctgtgtgt ctctgccaat ctgctgggct ggtccctctc atttttaacca 54000 gtctgagtga caggtcccct tcgctcatca ttcagatggc tttccagatg 54050 accaggacga gtgggatatt ttgcccccaa cttggctcgg catgtgaatt 54100 cttagctctg caaggtgttt atgcctttgc gggtttcttg atgtgttcgc 54150 agtgtcaccc cagagtcaga actgtacaca tcccaaaatt tggtggccgt 54200 ggaacacatt cccggtgata gaattgctaa attgtcgtga aataggttag 54250 aatttttctt taaattatgg ttttcttatt cgtgaaaatt cggagagtgc 54300 tgctaaaatt ggattggtgt gatctttttg gtagttgtaa tttaacagaa 54350 aaaacacaaaa tttcaaccat tcttaatgtt acgtcctccc cccaccccct 54400 tctttcagtg gtatgcaacc actgcaatca ctgtgcatat gtcttttctt 54450 agcaaaagga ttttaaaact tgagccctgg accttttgtc ctatgtgtgt 54500 ggattccagg gcaactctag catcagagca aaagccttgg gtttctcgca 54550 ttcagtggcc tatctccaga ttgtctgatt tctgaatgta aagttgttgt 54600 gttttttttt aaatagtagt ttgtagtatt ttaaagaaag aacagatcga 54650 gttctaatta tgatctagct tgattttgtg ttgatccaaa tttgcatagc 54700 tgtttaatgt taagtcatga caatttattt ttcttggcat gctatgtaaa 54750 cttgaatttc ctatgtattt ttaattgtggt gttttaaata tggggaggggg 54800 tattgagcat tttttaggga gaaaaataaa tatatgctgt agtggccaca 54850 aataggccta tgatttagct ggcaggccag gttttctcaa gagcaaaatc 54900 accctctggc cccttggcag gtaaggcctc ccggtcagca ttatcctgcc 54950 agacctcggg gaggatacct gggagacaga agcctctgca cctactgtgc 55000 agaactctcc acttccccaa ccctccccag gtgggcaggg cggagggagc 55050 ctcagcctcc ttagactgac ccctcaggcc cctaggctgg ggggttgtaa 55100 ataacagcag tcaggttgtt taccagccct ttgcacctcc ccaggcagag 55150 ggagcctctg ttctggtggg ggccacctcc ctcagaggct ctgctagcca 55200 cactccgtgg cccacccttt gttaccagtt cttcctcctt cctcttttcc 55250 cctgcctttc tcattccttc cttcgtctcc ctttttgttc ctttgcctct 55300 tgcctgtccc ctaaaacttg actgtggcac tcagggtcaa acagactatc 55350 cattccccag catgaatgtg ccttttaatt agtgatctag aaagaagttc 55400 agccgaaccc acaccccaac tccctcccaa gaacttcggt gcctaaagcc 55450 tcctgttcca cctcaggttt tcacaggtgc tcccaccccca gttgaggctc 55500 ccacccacag ggctgtctgt cacaaaccca cctctgttgg gagctattga 55550 gccacctggg atgagatgac acaaggcact cctaccactg agcgcctttg 55600 ccaggtccag cctgggctca ggttccaaga ctcagctgcc taatcccagg 55650 gttgagcctt gtgctcgtgg cggaccccaa accactgccc tcctgggtac 55700 cagccctcag tgtggaggct gagctggtgc ctggccccag tcttatctgt 55750 gcctttactg ctttgcgcat ctcagatgct aacttggttc tttttccaga 55800 agcctttgta ttggttaaaa attattttcc attgcagaag cagctggact 55850 atgcaaaaag tatttctctg tcagttcccc actctatacc aaggatatta 55900 ttaaaactag aaatgactgc attgagaggg agttgtggga aataagaaga 55950 atgaaagcct ctctttctgt ccgcagatcc tgacttttcc aaagtgcctt 56000 aaaagaaatc agacaaatgc cctgagtggt aacttctgtg ttattttact 56050 cttaaaacca aactctacct tttcttgttg ttttttttt tttttttttt 56100 ttttttttgg ttaccttctc attcatgtca agtatgtggt tcattcttag 56150 aaccaaggga aatactgctc cccccatttg ctgacgtagt gctctcatgg 56200 gctcacctgg gcccaaggca cagccagggc acagttaggc ctggatgttt 56250 gcctggtccg tgagatgccg cgggtcctgt ttccttactg gggatttcag 56300 ggctgggggt tcagggagca tttccttttc ctgggagtta tgaccgcgaa 56350 gttgtcatgt gccgtgccct tttctgtttc tgtgtatcct attgctggtg 56400 actctgtgtg aactggcctt tgggaaagat cagagagggc agaggtggca 56450 caggacagta aaggagatgc tgtgctggcc ttcagcctgg acagggtctc 56500 tgctgactgc caggggcggg ggctctgcat agccaggatg acggctttca 56550 tgtcccagag acctgttgtg ctgtgtattt tgatttcctg tgtatgcaaa 56600 tgtgtgtatt taccattgtg tagggggctg tgtctgatct tggtgttcaa 56650 aacagaactg tatttttgcc tttaaaatta aataatataa cgtgaataaa 56700 tgaccctatc tttgtaac 56718 <210> 3 <211> 4214 <212> DNA <213> Homo sapiens <220> <223> wild-type B4GALT1 mRNA sequence <400> 3 gcgccucggg cggcuucucg ccgcucccag gucuggcugg cuggaggagu 50 cucagcucuc agccgcucgc ccgccccccgc uccgggcccu ccccuagucg 100 ccgcugguggg gcagcgccug gcgggcggcc cgcgggcggg ucgccucccc 150 uccuguagcc cacacccuuc uuaaagcggc ggcgggaaga ugaggcuucg 200 ggagccgcuc cugagcggca gcgccgcgau gccaggcgcg ucccuacagc 250 gggccugccg ccugcucgug gccgucugcg cucugcaccu uggcgucacc 300 cucguuuacu accuggcugg ccgcgaccug agccgccugc cccaacuggu 350 cggagucucc acaccgcugc agggcggcuc gaacagugcc gccgccaucg 400 ggcaguccuc cggggagcuc cggaccggag gggcccggcc gccgccuccu 450 cuaggcgccu ccucccagcc gcgcccgggu ggcgacucca gcccagucgu 500 ggauucuggc ccuggccccg cuagcaacuu gaccucgguc ccagugcccc 550 acaccaccgc acugucgcug cccgccugcc cugaggaguc cccgcugcuu 600 gugggcccca ugcugauuga guuuaacaug ccuguggacc uggagcucgu 650 ggcaaagcag aacccaaaug ugaagauggg cggccgcuau gcccccaggg 700 acugcgucuc uccucacaag guggccauca ucauuccauu ccgcaaccgg 750 caggagcacc ucaaguacug gcuauauuau uugcacccag uccugcagcg 800 ccagcagcug gacuauggca ucuauguuau caaccaggcg ggagacacua 850 uauucaaucg ugcuaagcuc cucaauguug gcuuucaaga agccuugaag 900 gacuaugacu acaccugcuu uguguuuagu gacguggacc ucauuccaau 950 gaaugaccau aaugcguaca ggguuuuuc acagccacgg cacauuuccg 1000 uugcaaugga uaaguuugga uucagccuac cuuauguuca guauuuugga 1050 ggugucucug cucuaaguaa acaacaguuu cuaaccauca auggauuucc 1100 uaauaauuau uggggcuggg gaggagaaga ugaugacauu uuuaacagau 1150 uaguuuuuag aggcaugucu auaucucgcc caaaugcugu ggucgggagg 1200 ugucgcauga uccgccacuc aagagacaag aaaaaugaac ccaauccuca 1250 gagguuugac cgaauugcac acacaaagga gacaaugcuc ucugaugguu 1300 ugaacucacu caccuaccag gugcuggaug uacagagaua cccauuguau 1350 acccaaauca caguggacau cgggacaccg agcuagcguu uugguacacg 1400 gauaagagac cugaaauuag ccagggaccu cugcugugug ucucugccaa 1450 ucugcugggc uggucccucu cauuuuuacc agucugagug acaggucccc 1500 uucgcucauc auucagaugg cuuuccagau gaccaggacg agugggauau 1550 uuugccccca acuuggcucg gcaugugaau ucuuagcucu gcaagguguu 1600 uaugccuuug cggguuucuu gauguguucg cagugucacc ccagagucag 1650 aacuguacac aucccaaaau uugguggccg uggaacacau ucccggugau 1700 agaauugcua aauugucgug aaauagguua gaauuuuucu uuaaauuaug 1750 guuuucuuau ucgugaaaau ucggagagug cugcuaaaau uggauuggug 1800 ugaucuuuuu gguaguugua auuuaacaga aaaacacaaa auuucaacca 1850 uucuuaaugu uacguccucc ccccaccccc uucuuucagu gguaugcaac 1900 cacugcaauc acugugcaua ugucuuuucu uagcaaaagg auuuuaaaac 1950 uugagcccug gaccuuuugu ccuaugugug uggauuccag ggcaacucua 2000 gcaucagagc aaaagccuug gguuucucgc auucaguggc cuaucuccag 2050 auugucugau uucugaaugu aaaguuguug uguuuuuuuu uaaauguag 2100 uuuguaguau uuuaaagaaa gaacagaucg aguucuaauu augaucuagc 2150 uugauuuugu guugauccaa auuugcauag cuguuuaaug uuaagucaug 2200 acaauuuauu uuucuuggca ugcuauguaa acuugaauuu ccuauguauu 2250 uuuauuggg uguuuuaaau auggggaggg guauugagca uuuuuuaggg 2300 agaaaaauaa auauaugcug uaguggccac aaauaggccu augauuuagc 2350 uggcaggcca gguuuucuca agagcaaaau cacccucugg ccccuuggca 2400 gguaaggccu cccggucagc auuauccugc cagaccucgg ggaggauacc 2450 ugggagacag aagccucugc accuacugug cagaacucuc cacuucccca 2500 acccuccca ggugggcagg gcggagggag ccucagccuc cuuagacuga 2550 ccccucaggc cccuaggcug ggggguugua aauaacagca gucagguugu 2600 uuaccagccc uuugcaccuc cccaggcaga gggagccucu guucuggugg 2650 gggccaccuc ccucagaggc ucugcuagcc acacuccgug gcccacccuu 2700 uguuaccagu ucuuccuccu uccucuuuuc cccugccuuu cucauuccuu 2750 ccuucgucuc ccuuuuuguu ccuuugccuc uugccugucc ccuaaaacuu 2800 gacuguggca cucaggguca aacagacuau ccauucccca gcaugaaugu 2850 gccuuuuaau uagugaucua gaaagaaguu cagccgaacc cacaccccaa 2900 cucccuccca agaacuucgg ugccuaaagc cuccuguucc accucagguu 2950 uucacaggg cucccacccc aguugaggcu cccacccaca gggcugucug 3000 ucacaaaccc accucuguug ggagcuauug agccaccugg gaugagauga 3050 cacaaggcac uccuaccacu gagcgccuuu gccagggucca gccugggcuc 3100 agguuccaag acucagcugc cuaaucccag gguugagccu ugugcucgug 3150 gcggacccca aaccacugcc cuccugggua ccagcccuca guguggaggc 3200 ugagcuggug ccuggcccca gucuuaucug ugccuuuacu gcuuugcgca 3250 ucucagaugc uaacuugguu cuuuuuccag aagccuuugu auugguuaaa 3300 aauuauuuuc cauugcagaa gcagcuggac uaugcaaaaa guauuucucu 3350 gucaguuccc cacucuauac caaggauauu auuaaaacua gaaaugacug 3400 cauugagagg gaguugguggg aaauaagaag aaugaaagcc ucucuuucug 3450 uccgcagauc cugacuuuuc caaagugccu uaaaagaaau cagacaaaug 3500 cccugagugg uaacuucugu guuauuuuac ucuuaaaacc aaacucuuacc 3550 uuuucuuguu guuuuuuuuu uuuuuuuuuu uuuuuuuuug guuaccuucu 3600 cauucauguc aaguauggg uucaucuua gaaccaaggg aaauacugcu 3650 ccccccauuu gcugacguag ugcucucaug ggcucaccug ggcccaaggc 3700 acagccaggg cacaguuagg ccuggauguu ugccuggucc gugagaugcc 3750 gcggguccug uuuccuuacu ggggauuuca gggcuggggg uucagggagc 3800 auuuccuuuu ccugggaguu augaccgcga aguugucaug ugccgugccc 3850 uuuucuguuu cuguguaucc uauugcuggu gacucugugu gaacuggccu 3900 uugggaaaga ucagagaggg cagagguggc acaggacagu aaaggagaug 3950 cugugcuggc cuucagccug gacagggucu cugcugacug ccaggggcgg 4000 gggcucugca uagccaggau gacggcuuuc augucccaga gaccuguugu 4050 gcuguguauu uugauuuccu guguaugcaa auguguguau uuaccauugu 4100 guaggggggcu gugucugauc uugguguuca aaacagaacu guauuuuugc 4150 cuuuaaaauu aaauaauaua acgugaauaa augacccuau cuuuguaaca 4200 aaaaaaaaaaaaaa 4214 <210> 4 <211> 4214 <212> DNA <213> Homo sapiens <220> <223> variant B4GALT1 mRNA sequence <400> 4 gcgccucggg cggcuucucg ccgcucccag gucuggcugg cuggaggagu 50 cucagcucuc agccgcucgc ccgccccccgc uccgggcccu ccccuagucg 100 ccgcugguggg gcagcgccug gcgggcggcc cgcgggcggg ucgccucccc 150 uccuguagcc cacacccuuc uuaaagcggc ggcgggaaga ugaggcuucg 200 ggagccgcuc cugagcggca gcgccgcgau gccaggcgcg ucccuacagc 250 gggccugccg ccugcucgug gccgucugcg cucugcaccu uggcgucacc 300 cucguuuacu accuggcugg ccgcgaccug agccgccugc cccaacuggu 350 cggagucucc acaccgcugc agggcggcuc gaacagugcc gccgccaucg 400 ggcaguccuc cggggagcuc cggaccggag gggcccggcc gccgccuccu 450 cuaggcgccu ccucccagcc gcgcccgggu ggcgacucca gcccagucgu 500 ggauucuggc ccuggccccg cuagcaacuu gaccucgguc ccagugcccc 550 acaccaccgc acugucgcug cccgccugcc cugaggaguc cccgcugcuu 600 gugggcccca ugcugauuga guuuaacaug ccuguggacc uggagcucgu 650 ggcaaagcag aacccaaaug ugaagauggg cggccgcuau gcccccaggg 700 acugcgucuc uccucacaag guggccauca ucauuccauu ccgcaaccgg 750 caggagcacc ucaaguacug gcuauauuau uugcacccag uccugcagcg 800 ccagcagcug gacuauggca ucuauguuau caaccaggcg ggagacacua 850 uauucaaucg ugcuaagcuc cucaauguug gcuuucaaga agccuugaag 900 gacuaugacu acaccugcuu uguguuuagu gacguggacc ucauuccaau 950 gaaugaccau aaugcguaca ggguuuuuc acagccacgg cacauuuccg 1000 uugcaaugga uaaguuugga uucagccuac cuuauguuca guauuuugga 1050 ggugucucug cucuaaguaa acaacaguuu cuaaccauca auggauuucc 1100 uaauaauuau uggggcuggg gaggagaaga ugaugacauu uuuaacagau 1150 uaguuuuuag aggcaugucu auaucucgcc caaaugcugu ggucgggagg 1200 ugucgcauga uccgccacuc aagagacaag aaaaaugaac ccaguccuca 1250 gagguuugac cgaauugcac acacaaagga gacaaugcuc ucugaugguu 1300 ugaacucacu caccuaccag gugcuggaug uacagagaua cccauuguau 1350 acccaaauca caguggacau cgggacaccg agcuagcguu uugguacacg 1400 gauaagagac cugaaauuag ccagggaccu cugcugugug ucucugccaa 1450 ucugcugggc uggucccucu cauuuuuacc agucugagug acaggucccc 1500 uucgcucauc auucagaugg cuuuccagau gaccaggacg agugggauau 1550 uuugccccca acuuggcucg gcaugugaau ucuuagcucu gcaagguguu 1600 uaugccuuug cggguuucuu gauguguucg cagugucacc ccagagucag 1650 aacuguacac aucccaaaau uugguggccg uggaacacau ucccggugau 1700 agaauugcua aauugucgug aaauagguua gaauuuuucu uuaaauuaug 1750 guuuucuuau ucgugaaaau ucggagagug cugcuaaaau uggauuggug 1800 ugaucuuuuu gguaguugua auuuaacaga aaaacacaaa auuucaacca 1850 uucuuaaugu uacguccucc ccccaccccc uucuuucagu gguaugcaac 1900 cacugcaauc acugugcaua ugucuuuucu uagcaaaagg auuuuaaaac 1950 uugagcccug gaccuuuugu ccuaugugug uggauuccag ggcaacucua 2000 gcaucagagc aaaagccuug gguuucucgc auucaguggc cuaucuccag 2050 auugucugau uucugaaugu aaaguuguug uguuuuuuuu uaaauguag 2100 uuuguaguau uuuaaagaaa gaacagaucg aguucuaauu augaucuagc 2150 uugauuuugu guugauccaa auuugcauag cuguuuaaug uuaagucaug 2200 acaauuuauu uuucuuggca ugcuauguaa acuugaauuu ccuauguauu 2250 uuuauuggg uguuuuaaau auggggaggg guauugagca uuuuuuaggg 2300 agaaaaauaa auauaugcug uaguggccac aaauaggccu augauuuagc 2350 uggcaggcca gguuuucuca agagcaaaau cacccucugg ccccuuggca 2400 gguaaggccu cccggucagc auuauccugc cagaccucgg ggaggauacc 2450 ugggagacag aagccucugc accuacugug cagaacucuc cacuucccca 2500 acccuccca ggugggcagg gcggagggag ccucagccuc cuuagacuga 2550 ccccucaggc cccuaggcug ggggguugua aauaacagca gucagguugu 2600 uuaccagccc uuugcaccuc cccaggcaga gggagccucu guucuggugg 2650 gggccaccuc ccucagaggc ucugcuagcc acacuccgug gcccacccuu 2700 uguuaccagu ucuuccuccu uccucuuuuc cccugccuuu cucauuccuu 2750 ccuucgucuc ccuuuuuguu ccuuugccuc uugccugucc ccuaaaacuu 2800 gacuguggca cucaggguca aacagacuau ccauucccca gcaugaaugu 2850 gccuuuuaau uagugaucua gaaagaaguu cagccgaacc cacaccccaa 2900 cucccuccca agaacuucgg ugccuaaagc cuccuguucc accucagguu 2950 uucacaggg cucccacccc aguugaggcu cccacccaca gggcugucug 3000 ucacaaaccc accucuguug ggagcuauug agccaccugg gaugagauga 3050 cacaaggcac uccuaccacu gagcgccuuu gccagggucca gccugggcuc 3100 agguuccaag acucagcugc cuaaucccag gguugagccu ugugcucgug 3150 gcggacccca aaccacugcc cuccugggua ccagcccuca gugguggaggc 3200 ugagcuggug ccuggcccca gucuuaucug ugccuuuacu gcuuugcgca 3250 ucucagaugc uaacuugguu cuuuuuccag aagccuuugu auugguuaaa 3300 aauuauuuuc cauugcagaa gcagcuggac uaugcaaaaa guauuucucu 3350 gucaguuccc cacucuauac caaggauauu auuaaaacua gaaaugacug 3400 cauugagagg gaguugggg aaauaagaag aaugaaagcc ucucuuucug 3450 uccgcagauc cugacuuuuc caaagugccu uaaaagaaau cagacaaaug 3500 cccugagugg uaacuucugu guuauuuuac ucuuaaaacc aaacucuuacc 3550 uuuucuuguu guuuuuuuuu uuuuuuuuuu uuuuuuuuug guuaccuucu 3600 cauucauguc aaguauggg uucaucuua gaaccaaggg aaauacugcu 3650 ccccccauuu gcugacguag ugcucucaug ggcucaccug ggcccaaggc 3700 acagccaggg cacaguuagg ccuggauguu ugccuggucc gugagaugcc 3750 gcggguccug uuuccuuacu ggggauuuca gggcuggggg uucagggagc 3800 auuuccuuuu ccugggaguu augaccgcga aguugucaug ugccgugccc 3850 uuuucuguuu cuguguaucc uauugcuggu gacucugugu gaacuggccu 3900 uugggaaaga ucagagaggg cagagguggc acaggacagu aaaggagaug 3950 cugugcuggc cuucagccug gacagggucu cugcugacug ccaggggcgg 4000 gggcucugca uagccaggau gacggcuuuc augucccaga gaccuguugu 4050 gcuguguauu uugauuuccu guguaugcaa auguguguau uuaccauugu 4100 guaggggggcu gugucugauc uugguguuca aaacagaacu guauuuuugc 4150 cuuuaaaauu aaauaauaua acgugaauaa augacccuau cuuuguaaca 4200 aaaaaaaaaaaaaa 4214 <210> 5 <211> 1197 <212> DNA <213> Homo sapiens <220> <223> wild-type B4GALT1 cDNA sequence <400> 5 atgaggcttc gggagccgct cctgagcggc agcgccgcga tgccaggcgc 50 gtccctacag cgggcctgcc gcctgctcgt ggccgtctgc gctctgcacc 100 ttggcgtcac cctcgtttac tacctggctg gccgcgacct gagccgcctg 150 ccccaactgg tcggagtctc cacaccgctg cagggcggct cgaacagtgc 200 cgccgccatc gggcagtcct ccggggagct ccggaccgga ggggcccggc 250 cgccgcctcc tctaggcgcc tcctcccagc cgcgcccggg tggcgactcc 300 agcccagtcg tggattctgg ccctggcccc gctagcaact tgacctcggt 350 cccagtgccc cacaccaccg cactgtcgct gcccgcctgc cctgaggagt 400 ccccgctgct tgtgggcccc atgctgattg agtttaacat gcctgtggac 450 ctggagctcg tggcaaagca gaacccaaat gtgaagatgg gcggccgcta 500 tgcccccagg gactgcgtct ctcctcacaa ggtggccatc atcattccat 550 tccgcaaccg gcaggagcac ctcaagtact ggctatatta tttgcaccca 600 gtcctgcagc gccagcagct ggactatggc atctatgtta tcaaccaggc 650 gggagacact atattcaatc gtgctaagct cctcaatgtt ggctttcaag 700 aagccttgaa ggactatgac tacacctgct ttgtgtttag tgacgtggac 750 ctcattccaa tgaatgacca taatgcgtac aggtgttttt cacagccacg 800 gcacatttcc gttgcaatgg ataagtttgg attcagccta ccttatgttc 850 agtattttgg aggtgtctct gctctaagta aacaacagtt tctaaccatc 900 aatggatttc ctaataatta ttggggctgg ggaggagaag atgatgacat 950 ttttaacaga ttagttttta gaggcatgtc tatatctcgc ccaaatgctg 1000 tggtcggggag gtgtcgcatg atccgccact caagagacaa gaaaaaatgaa 1050 cccaatcctc agaggtttga ccgaattgca cacacaaagg agacaatgct 1100 ctctgatggt ttgaactcac tcacctacca ggtgctggat gtacagagat 1150 acccattgta tacccaaatc acagtggaca tcgggacacc gagctag 1197 <210> 6 <211> 1197 <212> DNA <213> Homo sapiens <220> <223> variant B4GALT1 cDNA sequence <400> 6 atgaggcttc gggagccgct cctgagcggc agcgccgcga tgccaggcgc 50 gtccctacag cgggcctgcc gcctgctcgt ggccgtctgc gctctgcacc 100 ttggcgtcac cctcgtttac tacctggctg gccgcgacct gagccgcctg 150 ccccaactgg tcggagtctc cacaccgctg cagggcggct cgaacagtgc 200 cgccgccatc gggcagtcct ccggggagct ccggaccgga ggggcccggc 250 cgccgcctcc tctaggcgcc tcctcccagc cgcgcccggg tggcgactcc 300 agcccagtcg tggattctgg ccctggcccc gctagcaact tgacctcggt 350 cccagtgccc cacaccaccg cactgtcgct gcccgcctgc cctgaggagt 400 ccccgctgct tgtgggcccc atgctgattg agtttaacat gcctgtggac 450 ctggagctcg tggcaaagca gaacccaaat gtgaagatgg gcggccgcta 500 tgcccccagg gactgcgtct ctcctcacaa ggtggccatc atcattccat 550 tccgcaaccg gcaggagcac ctcaagtact ggctatatta tttgcaccca 600 gtcctgcagc gccagcagct ggactatggc atctatgtta tcaaccaggc 650 gggagacact atattcaatc gtgctaagct cctcaatgtt ggctttcaag 700 aagccttgaa ggactatgac tacacctgct ttgtgtttag tgacgtggac 750 ctcattccaa tgaatgacca taatgcgtac aggtgttttt cacagccacg 800 gcacatttcc gttgcaatgg ataagtttgg attcagccta ccttatgttc 850 agtattttgg aggtgtctct gctctaagta aacaacagtt tctaaccatc 900 aatggatttc ctaataatta ttggggctgg ggaggagaag atgatgacat 950 ttttaacaga ttagttttta gaggcatgtc tatatctcgc ccaaatgctg 1000 tggtcggggag gtgtcgcatg atccgccact caagagacaa gaaaaaatgaa 1050 cccagtcctc agaggtttga ccgaattgca cacacaaagg agacaatgct 1100 ctctgatggt ttgaactcac tcacctacca ggtgctggat gtacagagat 1150 acccattgta tacccaaatc acagtggaca tcgggacacc gagctag 1197 <210> 7 <211> 398 <212> PRT <213> Homo sapiens <220> <223> wild-type B4GALT1 sequence <400> 7 Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ser Ala Ala Met Pro Gly 1 5 10 15 Ala Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala Val Cys Ala Leu 20 25 30 His Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly Arg Asp Leu Ser 35 40 45 Arg Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly Ser 50 55 60 Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu Leu Arg Thr Gly 65 70 75 80 Gly Ala Arg Pro Pro Pro Pro Pro Leu Gly Ala Ser Ser Gln Pro Arg Pro 85 90 95 Gly Gly Asp Ser Ser Pro Val Val Asp Ser Gly Pro Gly Pro Ala Ser 100 105 110 Asn Leu Thr Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro 115 120 125 Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met Leu Ile Glu 130 135 140 Phe Asn Met Pro Val Asp Leu Glu Leu Val Ala Lys Gln Asn Pro Asn 145 150 155 160 Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser Pro His 165 170 175 Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys 180 185 190 Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln Leu Asp 195 200 205 Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr Ile Phe Asn Arg 210 215 220 Ala Lys Leu Leu Asn Val Gly Phe Gln Glu Ala Leu Lys Asp Tyr Asp 225 230 235 240 Tyr Thr Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp 245 250 255 His Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala 260 265 270 Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr Phe Gly Gly 275 280 285 Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly Phe Pro 290 295 300 Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg 305 310 315 320 Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly 325 330 335 Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro Asn 340 345 350 Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr Met Leu Ser 355 360 365 Asp Gly Leu Asn Ser Leu Thr Tyr Gln Val Leu Asp Val Gln Arg Tyr 370 375 380 Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile Gly Thr Pro Ser 385 390 395 <210> 8 <211> 398 <212> PRT <213> Homo sapiens <220> <223> variant B4GALT1 sequence <400> 8 Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ser Ala Ala Met Pro Gly 1 5 10 15 Ala Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala Val Cys Ala Leu 20 25 30 His Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly Arg Asp Leu Ser 35 40 45 Arg Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly Ser 50 55 60 Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu Leu Arg Thr Gly 65 70 75 80 Gly Ala Arg Pro Pro Pro Pro Pro Leu Gly Ala Ser Ser Gln Pro Arg Pro 85 90 95 Gly Gly Asp Ser Ser Pro Val Val Asp Ser Gly Pro Gly Pro Ala Ser 100 105 110 Asn Leu Thr Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro 115 120 125 Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met Leu Ile Glu 130 135 140 Phe Asn Met Pro Val Asp Leu Glu Leu Val Ala Lys Gln Asn Pro Asn 145 150 155 160 Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser Pro His 165 170 175 Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys 180 185 190 Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln Leu Asp 195 200 205 Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr Ile Phe Asn Arg 210 215 220 Ala Lys Leu Leu Asn Val Gly Phe Gln Glu Ala Leu Lys Asp Tyr Asp 225 230 235 240 Tyr Thr Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp 245 250 255 His Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala 260 265 270 Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr Phe Gly Gly 275 280 285 Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly Phe Pro 290 295 300 Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg 305 310 315 320 Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly 325 330 335 Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro Ser 340 345 350 Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr Met Leu Ser 355 360 365 Asp Gly Leu Asn Ser Leu Thr Tyr Gln Val Leu Asp Val Gln Arg Tyr 370 375 380 Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile Gly Thr Pro Ser 385 390 395 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> guide RNA recognition sequences <400> 9 attagttttt agaggcatgt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> guide RNA recognition sequences <400> 10 ggctctcagg ccaagtgtat 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> guide RNA recognition sequences <400> 11 tactccttcc ccctttagga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> guide RNA recognition sequences <400> 12 gtccgaggct ctgggcctag 20 <210> 13 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> PAM for Cas9 from S. aureus <220> <221> n is A, G, C, or T <222> (1) .. (2) <220> <221> r is A or G <222> (4) .. (5) <400> 13 nngrrt 6 <210> 14 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> PAM for Cas9 from S. aureus <220> <221> n is A, G, C, or T <222> (1) .. (2) <220> <221> r is A or G <222> (4) .. (5) <400> 14 nngrr 5 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> target motif preceding NGG recognized by Cas9 protein <220> <221> n is A, G, C, or T <222> (2) .. (21) <400> 15 gnnnnnnnnnnnnnnnnnnnn ngg 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> target motif preceding NGG recognized by Cas9 protein <220> <221> n is A, G, C, or T <222> (1) .. (21) <400> 16 nnnnnnnnnn nnnnnnnnnn ngg 23 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RNA recognition sequence <220> <221> n is A, G, C, or T <222> (3) .. (23) <400> 17 ggnnnnnnnn nnnnnnnnnn nnngg 25

Claims (215)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete An isolated nucleic acid molecule or a complement thereof comprising a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 8 and encoding a polypeptide having the activity of beta-1,4-galactosyltransferase 1 (B4GALT1), provided that the polypeptide is numbered 352 An isolated nucleic acid molecule or complement thereof comprising a serine at position. According to clause 34,
An isolated nucleic acid molecule or complement thereof, wherein the nucleic acid sequence encodes the polypeptide sequence of SEQ ID NO: 8. A vector comprising an isolated nucleic acid molecule according to claim 34 or its complement. delete delete delete An isolated nucleic acid molecule according to claim 34 or 35 or its complement, or a vector according to claim 36 and
A composition comprising a carrier. delete A host cell comprising an isolated nucleic acid molecule according to claim 34 or 35 or its complement, or a vector according to claim 36. delete delete delete delete delete delete encoding human beta-1,4-galactosyltransferase 1 ( B4GALT1 ) protein comprising a nucleic acid sequence at least 99% identical to SEQ ID NO:6 As cDNA or its complement,
Provided that the nucleic acid sequence encodes a serine at a position corresponding to position 352 of the full-length or mature B4GALT1 polypeptide,
The cDNA is derived from a nucleic acid sequence encoding the B4GALT1 variant polypeptide of SEQ ID NO: 8, or its complement. delete A vector comprising the cDNA or its complement according to claim 49. delete delete delete cDNA or its complement according to claim 49, or a vector according to claim 51 and
A composition comprising a carrier. delete A host cell comprising a cDNA according to claim 49 or its complement, or a vector according to claim 51. delete delete delete delete delete delete An isolated polypeptide comprising an amino acid sequence that is at least 99% identical to the B4GALT1 variant polypeptide having SEQ ID NO: 8 and having the activity of B4GALT1 (beta-1,4-galactosyltransferase 1),
However, the polypeptide contains serine corresponding to position 352 of SEQ ID NO: 8. delete delete delete delete delete delete A composition comprising the polypeptide according to claim 64 and a carrier or excipient. A host cell expressing the polypeptide according to claim 64. A method for producing a polypeptide according to claim 64, comprising:
A method for producing a polypeptide, comprising culturing a host cell containing a nucleic acid molecule encoding the polypeptide, causing the cell to express the polypeptide, and recovering the expressed polypeptide. delete delete A method of providing detection information for B4GALT1 variant nucleic acid molecules for use in determining the susceptibility of a human subject to the development of a cardiovascular condition, comprising:
A sample obtained from the subject is assayed to determine whether the nucleic acid molecule in the sample contains a nucleic acid sequence encoding a serine at a position corresponding to position 352 of the full-length or mature B4GALT1 polypeptide with an amino acid sequence of 398 contiguous amino acids. Including the step of determining whether,
The method wherein the B4GALT1 variant nucleic acid molecule encodes the B4GALT1 variant polypeptide of SEQ ID NO: 8. According to clause 76,
The test is,
Sequencing a portion of the B4GALT1 genomic sequence of a nucleic acid molecule in the sample, wherein the sequenced portion comprises positions corresponding to positions 53575 to 53577 of SEQ ID NO: 2;
sequencing a portion of the B4GALT1 mRNA sequence of a nucleic acid molecule in the sample, wherein the sequenced portion comprises positions corresponding to positions 1243 to 1245 of SEQ ID NO:4; or
Sequencing a portion of the B4GALT1 cDNA sequence of a nucleic acid molecule in the sample, wherein the sequenced portion comprises positions corresponding to positions 1054 to 1056 of SEQ ID NO:6.
Method, including. According to clause 76,
The test is,
a) the sample comprising: i) a portion of the B4GALT1 genomic sequence adjacent to the position of the B4GALT1 genomic sequence corresponding to positions 53575 to 53577 of SEQ ID NO: 2; ii) a portion of the B4GALT1 mRNA sequence adjacent to the position of B4GALT1 mRNA corresponding to positions 1243 to 1245 of SEQ ID NO: 4; or iii) contacting with a primer that hybridizes to a portion of the B4GALT1 cDNA sequence adjacent to the position of the B4GALT1 cDNA corresponding to positions 1054 to 1056 of SEQ ID NO:6;
b) apply the primers to at least i) a position in the B4GALT1 genome sequence corresponding to positions 53575 to 53577; ii) the position of the B4GALT1 mRNA corresponding to positions 1243 to 1245; or iii) extending through the position of the B4GALT1 cDNA corresponding to positions 1054 to 1056; and
c) the product of the extension of the primer encodes serine at position 352 of SEQ ID NO: 8; i) a position corresponding to positions 53575 to 53577 of the B4GALT1 genome sequence; ii) a position corresponding to positions 1243 to 1245 of the B4GALT1 mRNA; or iii) determining whether it contains a nucleotide at a position corresponding to positions 1054 to 1056 of the B4GALT1 cDNA.
Method, including. According to clause 76,
The assay involves contacting the sample under stringent conditions with primers or probes that specifically hybridize to the B4GALT1 variant genomic sequence, mRNA sequence, or cDNA sequence rather than the corresponding wild-type B4GALT1 sequence and determining whether hybridization has occurred. A method comprising steps. A method of providing detection information for the presence of B4GALT1 Asn352Ser for use in determining the susceptibility of a human subject to the development of a cardiovascular condition, comprising:
Performing an assay on a sample obtained from the human subject to determine whether the B4GALT1 protein in the sample contains a serine residue at position 352,
The method wherein the detection information is based on the B4GALT1 variant polypeptide of SEQ ID NO: 8. An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, comprising:
a) Assaying a sample obtained from the subject and determining that the nucleic acid molecule in the sample comprises a nucleic acid sequence encoding a serine at a position corresponding to position 352 of the full-length or mature B4GALT1 polypeptide having an amino acid sequence of 398 contiguous amino acids. deciding whether or not to do so; and
b) If the nucleic acid molecule comprises a nucleic acid sequence encoding a serine at a position corresponding to position 352 of the full-length or mature B4GALT1 polypeptide, the human subject is classified as having a reduced risk for developing the cardiovascular condition. Alternatively, if the nucleic acid molecule does not contain a nucleic acid sequence encoding a serine at a position corresponding to position 352 of the full-length/mature B4GALT1 polypeptide, the human subject is at increased risk for developing the cardiovascular condition. Steps to classify
Including,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein said determination is based on the B4GALT1 variant polypeptide of SEQ ID NO:8. According to clause 81,
The test is,
Sequencing a portion of the B4GALT1 genomic sequence of a nucleic acid molecule in the sample, wherein the sequenced portion comprises positions corresponding to positions 53575 to 53577 of SEQ ID NO: 2;
sequencing a portion of the B4GALT1 mRNA sequence of a nucleic acid molecule in the sample, wherein the sequenced portion comprises positions corresponding to positions 1243 to 1245 of SEQ ID NO:4; or
Sequencing a portion of the B4GALT1 cDNA sequence of a nucleic acid molecule in the sample, wherein the sequenced portion comprises positions corresponding to positions 1054 to 1056 of SEQ ID NO:6.
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, comprising: According to clause 81,
The test is,
a) the sample comprising: i) a portion of the B4GALT1 genomic sequence adjacent to the position of the B4GALT1 genomic sequence corresponding to positions 53575 to 53577 of SEQ ID NO: 2; ii) a portion of the B4GALT1 mRNA sequence adjacent to the position of B4GALT1 mRNA corresponding to positions 1243 to 1245 of SEQ ID NO: 4; or iii) contacting with a primer that hybridizes to a portion of the B4GALT1 cDNA sequence adjacent to the position of the B4GALT1 cDNA corresponding to positions 1054 to 1056 of SEQ ID NO:6;
b) apply the primers to at least i) a position in the B4GALT1 genome sequence corresponding to positions 53575 to 53577; ii) the position of the B4GALT1 mRNA corresponding to positions 1243 to 1245; or iii) extending through the position of the B4GALT1 cDNA corresponding to positions 1054 to 1056; and
c) the product of the extension of the primer encodes serine at position 352 of SEQ ID NO: 8; i) a position corresponding to positions 53575 to 53577 of the B4GALT1 genome sequence; ii) a position corresponding to positions 1243 to 1245 of the B4GALT1 mRNA; or iii) determining whether it contains a nucleotide at a position corresponding to positions 1054 to 1056 of the B4GALT1 cDNA.
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, comprising: According to clause 81,
The assay involves contacting the sample under stringent conditions with primers or probes that specifically hybridize to the B4GALT1 variant genomic sequence, mRNA sequence, or cDNA sequence rather than the corresponding wild-type B4GALT1 sequence and determining whether hybridization has occurred. An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, comprising the steps of: delete delete delete delete delete delete delete delete delete delete An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, comprising:
a) performing an assay on a sample obtained from the human subject to determine whether the B4GALT1 protein in the sample contains a serine residue at position 352; and
b) if the B4GALT1 polypeptide contains a serine at a position corresponding to position 352 of a full-length or mature B4GALT1 polypeptide with an amino acid sequence of 398 contiguous amino acids, then the human subject is at a reduced risk for developing the cardiovascular condition. or, if the B4GALT1 polypeptide does not contain a serine at the position corresponding to position 352 of the full-length or mature B4GALT1 polypeptide, the human subject is at increased risk for developing the cardiovascular condition. Steps to classify
Including,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein said determination is based on the B4GALT1 variant polypeptide of SEQ ID NO:8. The method according to any one of claims 81 to 84,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises increased levels of one or more serum lipids. According to clause 96,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the serum lipids include one or more of cholesterol, LDL, HDL, triglycerides, HDL-cholesterol, and non-HDL cholesterol. The method according to any one of claims 81 to 84,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises increased levels of coronary artery calcification. The method according to any one of claims 81 to 84,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises increased levels of pericardial fat. The method according to any one of claims 81 to 84,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition includes an atherothrombotic condition. According to clause 100,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the atherothrombotic condition comprises increased levels of fibrinogen. According to clause 101,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the atherothrombotic condition comprises a blood clot formed from involvement of fibrinogen activity. The method according to any one of claims 81 to 84,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises increased levels of fibrinogen. Paragraph 103:
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises a blood clot formed from involvement of fibrinogen activity. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete According to clause 95,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises increased levels of one or more serum lipids. According to clause 154,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the serum lipids include one or more of cholesterol, LDL, HDL, triglycerides, HDL-cholesterol, and non-HDL cholesterol. According to clause 95,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises increased levels of coronary artery calcification. According to clause 95,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises increased levels of pericardial fat. According to clause 95,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition includes an atherothrombotic condition. According to clause 158,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the atherothrombotic condition comprises increased levels of fibrinogen. Paragraph 159:
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the atherothrombotic condition comprises a blood clot formed from involvement of fibrinogen activity. According to clause 95,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises increased levels of fibrinogen. According to clause 161,
An in vitro method for determining the susceptibility of a human subject to the development of a cardiovascular condition, wherein the cardiovascular condition comprises a blood clot formed from involvement of fibrinogen activity.
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